Rab proteins

ABSTRACT

The invention provides three human Rab proteins (RABP) and polynucleotides which identify and encode RABP. The invention also provides expression vectors, host cells, agonists, antibodies and antagonists. The invention also provides methods for treating disorders associated with expression of RABP.

[0001] This application is a divisional application of U.S. applicationSer. No. 09/154,602, filed Sep. 16, 1998, which is a divisional of U.S.application Ser. No. 08/916,901, filed Aug. 21, 1997, issued Apr. 6,1999, as U.S. Pat. No. 5,892,012, entitled RAB PROTEINS, both of whichare hereby expressly incorporated by reference.

FIELD OF THE INVENTION

[0002] This invention relates to nucleic acid and amino acid sequencesof three new human Rab proteins and to the use of these sequences in thediagnosis, prevention, and treatment of inflammation and disordersassociated with cell proliferation and apoptosis.

BACKGROUND OF THE INVENTION

[0003] Vesicle trafficking is defined as the vesicular transport ofmaterials between different subcellular compartments of eukaryoticcells. Vesicles bud from a donor membrane and fuse with a recipient onecarrying internalized materials from one site to another. Rab proteins,low molecular weight (LMW) guanidine triphosphatases (GTPases), belongto the Ras superfamily; they help regulate vesicular transport bydirecting the vesicles to and from the correct membrane surfaces(Novick, P. and Brennwald, P. (1993) Cell 75: 597-601).

[0004] Rab proteins assist the binding of a transport vesicle to itsproper acceptor membrane and initiate the membrane fusion process usingthe energy derived from the hydrolysis of GTP. Rab proteins have ahighly variable amino terminus and a prenylated carboxy terminus. Theamino terminus contains signal sequences, and the carboxy terminusdetermines the target membrane to which the Rab protein binds. Thetargeting process is assisted by a series of escort proteins(Khosravi-Far, R. et al. (1991) Proc. Natl. Acad. Sci. 88: 6264-6268).

[0005] In the medial Golgi, it has been shown that GTP-bound Rabproteins initiate the binding of VAMP-like proteins of the transportvesicle to syntaxin-like proteins on the acceptor membrane, triggeringmembrane fusion events. After transport, GTPase-activating proteins inthe target membrane convert the Rab proteins to their GDP-bound state,and guanine-nucleotide dissociation inhibitor helps return the GDP-boundproteins to their membrane of origin.

[0006] To date, more than 30 Rab proteins have been identified, and eachmay have a characteristic intracellular location where it functions indistinct, tissue-specific transport events. For example, Rab2 isimportant in ER-to-Golgi transport; Rab1 and Rab6 are localized to theGolgi apparatus; Rab3 transports secretory vesicles to the extracellularmembrane; Rab5 and Rab7 are localized to the early and late endosomalfusion events, respectively; and Rab 10 mediates vesicle fusion from themedial Golgi to the trans Golgi.

[0007] Structurally, the Rab proteins display features characteristic ofLMW GTP-binding proteins. Four sequence regions, motifs I-IV, areconserved in the Rab proteins. Motif I, the most variable region amongthe four, has a signature of GXXXXGK, and the terminal lysine residueinteracts with the β- and γ-phosphates of GTP. Motifs II, III, and IVare highly conserved and function in regulating the binding ofγ-phosphate, GTP, and the guanine base of GTP, respectively.

[0008] In addition to the conserved motifs, the arginine residuefollowing the second GTP-binding domain, the phenylalanine residueadjacent to the fourth GTP-binding domain, and the carboxy terminalcysteines are highly conserved. The cysteines are particularly importantin that they are essential for membrane localization. The Rab proteinsalso have an effector region located in between Motif I and Motif IIwhich has been characterized as the interaction site for GAP, aregulatory protein which stimulates the intrinsic GTPase activity.

[0009] Experimental evidence has established the essential role of Rabin vesicle trafficking, cell function, and cell differentiation. HumanRab1, Rab2, Rab3B, Rab4, Rab5, and Rab6 genes isolated from a humanpheochromocytoma cDNA library exhibit GTPase activities when produced inE. coli (Zahraoui, A. et al. (1989) J. Biol. Chem. 264: 12394-12401).Although differentially expressed, two isoforms of Rab28, hRab28S andhRab28L, exhibit comparable GTPase-related activities in rat tissues(Brauers, A. et al. (1996) Eur. J. Biochem. 237: 833-840). Localizationof murine Rab24 in endoplasmic reticulum/cis-Golgi region of SemlikiForest virus and the vaccinia T7 vector systems indicates that Rab24 maybe involved in autophagy-related processes (Olkkonen, V. M. et al.(1993) J. Cell. Sci. 106: 1249-1261). Over expression of Rab proteinssignificantly enhances the function of Rev, a viral gene essential forprocessing HIV-1 (Fridell, R. A. et al. (1996) Proc. Natl. Acad. Sci.93: 4421-4424). A deficiency in the prenylation of one particular Rab isassociated with choroideremia, a form of retinal degeneration thatcauses blindness (Seabra, M. et al. (1996) J. Biol. Chem.270:24420-24427). Interaction between Rab protein and Cdc2 proteinkinase in vitro inhibited vesicle fusion and implicated Rab proteinfunction in mediating cell cycle events (Tuomikoski, T. et al. (1989,Nature 342: 942-945). Thus, Rab proteins appear to be involved in thecomplex and critical processes of vesicle trafficking for the directedrelease of various molecules.

[0010] The discovery of three new human Rab proteins and thepolynucleotides encoding them satisfies a need in the art by providingnew compositions which are useful in the diagnosis, prevention andtreatment of inflammation and disorders associated with cellproliferation and apoptosis.

SUMMARY OF THE INVENTION

[0011] The invention features three substantially purified polypeptides,designated individually as RABP-1, RABP-2 and RABP-3 and collectively asRABP having the amino acid sequences shown in SEQ ID NO:1, SEQ ID NO:3,and SEQ ID NO:5.

[0012] The invention further provides an isolated and substantiallypurified polynucleotide sequence encoding the polypeptide RAPB-1,comprising the amino acid sequence of SEQ ID NO:1 or fragments thereofand compositions comprising said polynucleotide sequence. The inventionalso provides a polynucleotide sequence which hybridizes under stringentconditions to the polynucleotide sequence encoding the amino acidsequence SEQ ID NO:1 or fragments of said polynucleotide sequence. Theinvention further provides a polynucleotide sequence comprising thecomplement of the polynucleotide sequence encoding the amino acidsequence of SEQ ID NO:1, or fragments or variants of said polynucleotidesequence.

[0013] The invention also provides an isolated and purified sequencecomprising SEQ ID NO:2, or variants thereof. In addition, the inventionprovides a polynucleotide sequence which hybridizes under stringentconditions to the polynucleotide sequence of SEQ ID NO:2. The inventionalso provides a polynucleotide sequence comprising the complement of SEQID NO:2, or fragments or variants thereof.

[0014] The present invention further provides an expression vectorcontaining at least a fragment of any of the claimed polynucleotidesequences. In yet another aspect, the expression vector containing thepolynucleotide sequence is contained within a host cell.

[0015] The invention also provides a method for producing a polypeptidecomprising the amino acid sequence of SEQ ID NO:1, or a fragmentthereof, the method comprising the steps of: a) culturing the host cellcontaining an expression vector containing at least a fragment of thepolynucleotide sequence encoding RABP-1 under conditions suitable forthe expression of the polypeptide; and b) recovering the polypeptidefrom the host cell culture.

[0016] The invention also provides a pharmaceutical compositioncomprising a substantially purified RABP-1 having the amino acidsequence of SEQ ID NO:1 in conjunction with a suitable pharmaceuticalcarrier.

[0017] The invention also provides a purified antagonist of thepolypeptide of SEQ ID NO:1. In one aspect the invention provides apurified antibody which binds to a polypeptide comprising the amino acidsequence of SEQ ID NO:1.

[0018] Still further, the invention provides a purified agonist of thepolypeptide of SEQ ID NO:1.

[0019] The invention also provides a method for stimulating cellproliferation comprising administering to a cell an effective amount ofa pharmaceutical composition comprising purified RABP-1.

[0020] The invention also provides a method for treating a disorderassociated with an increase in apoptosis comprising administering to asubject in need of such treatment an effective amount of apharmaceutical composition comprising purified RABP-1.

[0021] The invention also provides a method for treating cancercomprising administering to a subject in need of such treatment aneffective amount of an antagonist of RABP-1.

[0022] The invention also provides a method for treating inflammationcomprising administering to a subject in need of such treatment aneffective amount of an antagonist of RABP-1.

[0023] The invention also provides a method for detecting apolynucleotide which encodes RABP-1 in a biological sample comprisingthe steps of: a) hybridizing the complement of the polynucleotidesequence which encodes SEQ ID NO:1 to nucleic acid material of abiological sample, thereby forming a hybridization complex; and b)detecting the hybridization complex, wherein the presence of the complexcorrelates with the presence of a polynucleotide encoding RABP-1 in thebiological sample. In one aspect the nucleic acid material of thebiological sample is amplified by the polymerase chain reaction prior tohybridization.

[0024] Still further, the invention provides an isolated andsubstantially purified polynucleotide sequence encoding the polypeptideRABP-2, comprising the amino acid sequence of SEQ ID NO:3 or fragmentsthereof and compositions comprising said polynucleotide sequence. Theinvention also provides a polynucleotide sequence which hybridizes understringent conditions to the polynucleotide sequence encoding the aminoacid sequence SEQ ID NO:3 or fragments of said polynucleotide sequence.The invention further provides a polynucleotide sequence comprising thecomplement of the polynucleotide sequence encoding the amino acidsequence of SEQ ID NO:3, or fragments or variants of said polynucleotidesequence.

[0025] The invention also provides an isolated and purified sequencecomprising SEQ ID NO:4, or variants thereof. In addition, the inventionprovides a polynucleotide sequence which hybridizes under stringentconditions to the polynucleotide sequence of SEQ ID NO:4. The inventionalso provides a polynucleotide sequence comprising the complement of SEQID NO:4, or fragments or variants thereof.

[0026] The present invention further provides an expression vectorcontaining at least a fragment of any of the claimed polynucleotidesequences. In yet another aspect, the expression vector containing thepolynucleotide sequence is contained within a host cell.

[0027] The invention also provides a method for producing a polypeptidecomprising the amino acid sequence of SEQ ID NO:3, or a fragmentthereof, the method comprising the steps of: a) culturing the host cellcontaining an expression vector containing at least a fragment of thepolynucleotide sequence encoding RABP-2 under conditions suitable forthe expression of the polypeptide; and b) recovering the polypeptidefrom the host cell culture.

[0028] The invention also provides a pharmaceutical compositioncomprising a substantially purified RABP-2 having the amino acidsequence of SEQ ID NO:3 in conjunction with a suitable pharmaceuticalcarrier.

[0029] The invention also provides a purified antagonist of thepolypeptide of SEQ ID NO:3. In one aspect the invention provides apurified antibody which binds to a polypeptide comprising the amino acidsequence of SEQ ID NO:3.

[0030] Still further, the invention provides a purified agonist of thepolypeptide of SEQ ID NO:3.

[0031] The invention also provides a method for stimulating cellproliferation comprising administering to a cell an effective amount ofa pharmaceutical composition comprising purified RABP-2.

[0032] The invention also provides a method for treating a disorderassociated with an increase in apoptosis comprising administering to asubject in need of such treatment an effective amount of apharmaceutical composition comprising purified RABP-2.

[0033] The invention also provides a method for treating cancercomprising administering to a subject in need of such treatment aneffective amount of an antagonist of RABP-2.

[0034] The invention also provides a method for treating inflammationcomprising administering to a subject in need of such treatment aneffective amount of an antagonist of RABP-2.

[0035] The invention also provides a method for detecting apolynucleotide which encodes RABP-3 (SEQ ID NO:3) in a biological samplecomprising the steps of: a) hybridizing the complement of thepolynucleotide sequence which encodes SEQ ID NO:3 to nucleic acidmaterial of a biological sample, thereby forming a hybridizationcomplex; and b) detecting the hybridization complex, wherein thepresence of the complex correlates with the presence of a polynucleotideencoding RABP-2 in the biological sample. In one aspect the nucleic acidmaterial of the biological sample is amplified by the polymerase chainreaction prior to hybridization.

[0036] Still further, the invention provides an isolated andsubstantially purified polynucleotide sequence encoding the polypeptideRAPB-3, comprising the amino acid sequence of SEQ ID NO:5 or fragmentsthereof and compositions comprising said polynucleotide sequence. Theinvention also provides a polynucleotide sequence which hybridizes understringent conditions to the polynucleotide sequence encoding the aminoacid sequence SEQ ID NO:5 or fragments of said polynucleotide sequence.The invention further provides a polynucleotide sequence comprising thecomplement of the polynucleotide sequence encoding the amino acidsequence of SEQ ID NO:5, or fragments or variants of said polynucleotidesequence.

[0037] The invention also provides an isolated and purified sequencecomprising SEQ ID NO:6, or variants thereof. In addition, the inventionprovides a polynucleotide sequence which hybridizes under stringentconditions to the polynucleotide sequence of SEQ ID NO:6. The inventionalso provides a polynucleotide sequence comprising the complement of SEQID NO:6, or fragments or variants thereof.

[0038] The present invention further provides an expression vectorcontaining at least a fragment of any of the claimed polynucleotidesequences. In yet another aspect, the expression vector containing thepolynucleotide sequence is contained within a host cell.

[0039] The invention also provides a method for producing a polypeptidecomprising the amino acid sequence of SEQ ID NO:5, or a fragmentthereof, the method comprising the steps of: a) culturing the host cellcontaining an expression vector containing at least a fragment of thepolynucleotide sequence encoding RABP-3 under conditions suitable forthe expression of the polypeptide; and b) recovering the polypeptidefrom the host cell culture.

[0040] The invention also provides a pharmaceutical compositioncomprising a substantially purified RABP-3 having the amino acidsequence of SEQ ID NO:5 in conjunction with a suitable pharmaceuticalcarrier.

[0041] The invention also provides a purified antagonist of thepolypeptide of SEQ ID NO:5. In one aspect the invention provides apurified antibody which binds to a polypeptide comprising the amino acidsequence of SEQ ID NO:5.

[0042] Still further, the invention provides a purified agonist of thepolypeptide of SEQ ID NO:5.

[0043] The invention also provides a method for stimulating cellproliferation comprising administering to a cell an effective amount ofa pharmaceutical composition comprising purified RABP-3.

[0044] The invention also provides a method for treating a disorderassociated with an increase in apoptosis comprising administering to asubject in need of such treatment an effective amount of apharmaceutical composition comprising purified RABP-3.

[0045] The invention also provides a method for treating cancercomprising administering to a subject in need of such treatment aneffective amount of an antagonist of RABP-3.

[0046] The invention also provides a method for treating inflammationcomprising administering to a subject in need of such treatment aneffective amount of an antagonist of RABP-3.

[0047] The invention also provides a method for detecting apolynucleotide which encodes RABP-3 in a biological sample comprisingthe steps of: a) hybridizing the complement of the polynucleotidesequence which encodes SEQ ID NO:5 to nucleic acid material of abiological sample, thereby forming a hybridization complex; and b)detecting the hybridization complex, wherein the presence of the complexcorrelates with the presence of a polynucleotide encoding RABP-3 in thebiological sample. In one aspect the nucleic acid material of thebiological sample is amplified by the polymerase chain reaction prior tohybridization.

BRIEF DESCRIPTION OF THE FIGURES

[0048]FIGS. 1A, 1B, 1C, and 1D show the amino acid sequence (SEQ IDNO:1) and nucleic acid sequence (SEQ ID NO:2) of RABP-1. The alignmentwas produced using MACDNASIS PRO software (Hitachi Software EngineeringCo. Ltd. San Bruno, Calif.).

[0049]FIGS. 2A, 2B, and 2C show the amino acid sequence (SEQ ID NO:3)and nucleic acid sequence (SEQ ID NO:4) of RABP-2. The alignment wasproduced using MACDNASIS PRO software (Hitachi Software Engineering Co.Ltd. San Bruno, Calif.).

[0050]FIGS. 3A, 3B, 3C, 3D, 3E, 3F, 3G, and 3H show the amino acidsequence (SEQ ID NO:5) and nucleic acid sequence (SEQ ID NO:6) ofRABP-3. The alignment was produced using MACDNASIS PRO software (HitachiSoftware Engineering Co. Ltd. San Bruno, Calif.).

[0051]FIG. 4 shows the amino acid sequence alignments between RABP-1(2312652; SEQ ID NO:1) and a mouse Rab24 (GI 438164; SEQ ID NO:7),produced using the multisequence alignment program of DNASTAR software(DNASTAR Inc, Madison Wis.).

[0052]FIG. 5 shows the amino acid sequence alignments between RABP-2(2514506; SEQ ID NO:3) and a rat Rab1B (GI 57006; SEQ ID NO:8), producedusing the multisequence alignment program of DNAS TAR software (DNASTARInc, Madison Wis.).

[0053]FIG. 6 shows the amino acid sequence alignments between RABP-3(3400003; SEQ ID NO:5) and a rat Rab28 (GI 1154901; SEQ ID NO:9),produced using the multisequence alignment program of DNASTAR software(DNASTAR Inc, Madison Wis.).

[0054]FIGS. 7A and 7B show the hydrophobicity plots for RABP-1 (SEQ IDNO:1) and mouse Rab24 (SEQ ID NO:7), respectively. The positive X axisreflects amino acid position, and the negative Y axis, hydrophobicity(MACDNASIS PRO software).

[0055]FIGS. 8A and 8B show the hydrophobicity plots for RABP-2 (SEQ IDNO:3) and the rat Rab1B (SEQ ID NO:8), respectively. The positive X axisreflects amino acid position, and the negative Y axis, hydrophobicity(MACDNASIS PRO software).

[0056]FIGS. 9A and 9B show the hydrophobicity plots for RABP-3 (SEQ IDNO:5) and the rat Rab28 (SEQ ID NO:9), respectively. The positive X axisreflects amino acid position, and the negative Y axis, hydrophobicity(MACDNASIS PRO software).

DESCRIPTION OF THE INVENTION

[0057] Before the present proteins, nucleotide sequences, and methodsare described, it is understood that this invention is not limited tothe particular methodology, protocols, cell lines, vectors, and reagentsdescribed, as these may vary. It is also to be understood that theterminology used herein is for the purpose of describing particularembodiments only, and is not intended to limit the scope of the presentinvention which will be limited only by the appended claims.

[0058] It must be noted that as used herein and in the appended claims,the singular forms “a”, “an”, and “the” include plural reference unlessthe context clearly dictates otherwise. Thus, for example, reference to“a host cell” includes a plurality of such host cells, reference to the“antibody” is a reference to one or more antibodies and equivalentsthereof known to those skilled in the art, and so forth.

[0059] Unless defined otherwise, all technical and scientific terms usedherein have the same meanings as commonly understood by one of ordinaryskill in the art to which this invention belongs. Although any methodsand materials similar or equivalent to those described herein can beused in the practice or testing of the present invention, the preferredmethods, devices, and materials are now described. All publicationsmentioned herein are incorporated herein by reference for the purpose ofdescribing and disclosing the cell lines, vectors, and methodologieswhich are reported in the publications which might be used in connectionwith the invention. Nothing herein is to be construed as an admissionthat the invention is not entitled to antedate such disclosure by virtueof prior invention.

[0060] Definitions

[0061] RABP, as used herein, refers to the amino acid sequences ofsubstantially purified RABP obtained from any species, particularlymammalian, including bovine, ovine, porcine, murine, equine, andpreferably human, from any source whether natural, synthetic,semi-synthetic, or recombinant.

[0062] The term “agonist”, as used herein, refers to a molecule which,when bound to RABP, increases or prolongs the duration of the effect ofRABP. Agonists may include proteins, nucleic acids, carbohydrates, orany other molecules which bind to and modulate the effect of RABP.

[0063] An “allele” or “allelic sequence”, as used herein, is analternative form of the gene encoding RABP. Alleles may result from atleast one mutation in the nucleic acid sequence and may result inaltered mRNAs or polypeptides whose structure or function may or may notbe altered. Any given natural or recombinant gene may have none, one, ormany allelic forms. Common mutational changes which give rise to allelesare generally ascribed to natural deletions, additions, or substitutionsof nucleotides. Each of these types of changes may occur alone, or incombination with the others, one or more times in a given sequence.

[0064] “Altered” nucleic acid sequences encoding RABP, as used herein,include those with deletions, insertions, or substitutions of differentnucleotides resulting in a polynucleotide that encodes the same or afunctionally equivalent RABP. Included within this definition arepolymorphisms which may or may not be readily detectable using aparticular oligonucleotide probe of the polynucleotide encoding RABP,and improper or unexpected hybridization to alleles, with a locus otherthan the normal chromosomal locus for the polynucleotide sequenceencoding RABP. The encoded protein may also be “altered” and containdeletions, insertions, or substitutions of amino acid residues whichproduce a silent change and result in a functionally equivalent RABP.Deliberate amino acid substitutions may be made on the basis ofsimilarity in polarity, charge, solubility, hydrophobicity,hydrophilicity, and/or the amphipathic nature of the residues as long asthe biological or immunological activity of RABP is retained. Forexample, negatively charged amino acids may include aspartic acid andglutamic acid; positively charged amino acids may include lysine andarginine; and amino acids with uncharged polar head groups havingsimilar hydrophilicity values may include leucine, isoleucine, andvaline, glycine and alanine, asparagine and glutamine, serine andthreonine, and phenylalanine and tyrosine.

[0065] “Amino acid sequence”, as used herein, refers to an oligopeptide,peptide, polypeptide, or protein sequence, and fragment thereof, and tonaturally occurring or synthetic molecules. Fragments of RABP arepreferably about 5 to about 15 amino acids in length and retain thebiological activity or the immunological activity of RABP. Where “aminoacid sequence” is recited herein to refer to an amino acid sequence of anaturally occurring protein molecule, amino acid sequence, and liketerms, are not meant to limit the amino acid sequence to the complete,native amino acid sequence associated with the recited protein molecule.

[0066] “Amplification”, as used herein, refers to the production ofadditional copies of a nucleic acid sequence and is generally carriedout using polymerase chain reaction (PCR) technologies well known in theart (Dieffenbach, C. W. and G. S. Dveksler (1995) PCR Primer, aLaboratory Manual, Cold Spring Harbor Press, Plainview, N.Y.).

[0067] The term “antagonist”, as used herein, refers to a moleculewhich, when bound to RABP, decreases the amount or the duration of theeffect of the biological or immunological activity of RABP. Antagonistsmay include proteins, nucleic acids, carbohydrates, antibodies or anyother molecules which decrease the effect of RABP.

[0068] As used herein, the term “antibody” refers to intact molecules aswell as fragments thereof, such as Fa, F(ab′)₂, and Fv, which arecapable of binding the epitopic determinant. Antibodies that bind RABPpolypeptides can be prepared using intact polypeptides or fragmentscontaining small peptides of interest as the immunizing antigen. Thepolypeptide or oligopeptide used to immunize an animal can be derivedfrom the translation of RNA or synthesized chemically and can beconjugated to a carrier protein, if desired. Commonly used carriers thatare chemically coupled to peptides include bovine serum albumin andthyroglobulin, keyhole limpet hemocyanin. The coupled peptide is thenused to immunize the animal (e.g., a mouse, a rat, or a rabbit).

[0069] The term “antigenic determinant”, as used herein, refers to thatfragment of a molecule (i.e., an epitope) that makes contact with aparticular antibody. When a protein or fragment of a protein is used toimmunize a host animal, numerous regions of the protein may induce theproduction of antibodies which bind specifically to a given region orthree-dimensional structure on the protein; these regions or structuresare referred to as antigenic determinants. An antigenic determinant maycompete with the intact antigen (i.e., the immunogen used to elicit theimmune response) for binding to an antibody.

[0070] The term “antisense”, as used herein, refers to any compositioncontaining nucleotide sequences which are complementary to a specificDNA or RNA sequence. The term “antisense strand” is used in reference toa nucleic acid strand that is complementary to the “sense” strand.Antisense molecules include peptide nucleic acids and may be produced byany method including synthesis or transcription. Once introduced into acell, the complementary nucleotides combine with natural sequencesproduced by the cell to form duplexes and block either transcription ortranslation. The designation “negative” is sometimes used in referenceto the antisense strand, and “positive” is sometimes used in referenceto the sense strand.

[0071] The term “biologically active”, as used herein, refers to aprotein having structural, regulatory, or biochemical functions of anaturally occurring molecule. Likewise, “immunologically active” refersto the capability of the natural, recombinant, or synthetic RABP, or anyoligopeptide thereof, to induce a specific immune response inappropriate animals or cells and to bind with specific antibodies.

[0072] The terms “complementary” or “complementarity”, as used herein,refer to the natural binding of polynucleotides under permissive saltand temperature conditions by base-pairing. For example, the sequence“A-G-T” binds to the complementary sequence “T-C-A”. Complementaritybetween two single-stranded molecules may be “partial”, in which onlysome of the nucleic acids bind, or it may be complete when totalcomplementarity exists between the single stranded molecules. The degreeof complementarity between nucleic acid strands has significant effectson the efficiency and strength of hybridization between nucleic acidstrands. This is of particular importance in amplification reactions,which depend upon binding between nucleic acids strands and in thedesign and use of PNA molecules.

[0073] A “composition comprising a given polynucleotide sequence”, asused herein, refers broadly to any composition containing the givenpolynucleotide sequence. The composition may comprise a dry formulationor an aqueous solution. Compositions comprising polynucleotide sequencesencoding RABP (SEQ ID NO:1, SEQ ID NO:3, or SEQ ID NO:5) or fragmentsthereof (e.g., SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6 and fragmentsthereof) may be employed as hybridization probes. The probes may bestored in freeze-dried form and may be associated with a stabilizingagent such as a carbohydrate. In hybridizations, the probe may bedeployed in an aqueous solution containing salts (e.g., NaCl),detergents (e.g., SDS) and other components (e.g., Denhardt's solution,dry milk, salmon sperm DNA, etc.).

[0074] “Consensus”, as used herein, refers to a nucleic acid sequencewhich has been resequenced to resolve uncalled bases, has been extendedusing XL-PCR (Perkin Elmer, Norwalk, Conn.) in the 5′ and/or the 3′direction and resequenced, or has been assembled from the overlappingsequences of more than one Incyte Clone using a computer program forfragment assembly (e.g., GELVIEW Fragment Assembly system, GCG, Madison,Wis.). Some sequences have been both extended and assembled to producethe consensus sequence.

[0075] The term “correlates with expression of a polynucleotide”, asused herein, indicates that the detection of the presence of ribonucleicacid that is similar to SEQ ID NO:2, SEQ ID NO:4, or SEQ ID NO:6 bynorthern analysis is indicative of the presence of mRNA encoding RABP ina sample and thereby correlates with expression of the transcript fromthe polynucleotide encoding the protein.

[0076] A “deletion”, as used herein, refers to a change in the aminoacid or nucleotide sequence and results in the absence of one or moreamino acid residues or nucleotides.

[0077] The term “derivative”, as used herein, refers to the chemicalmodification of a nucleic acid encoding or complementary to RABP or theencoded RABP. Such modifications include, for example, replacement ofhydrogen by an alkyl, acyl, or amino group. A nucleic acid derivativeencodes a polypeptide which retains the biological or immunologicalfunction of the natural molecule. A derivative polypeptide is one whichis modified by glycosylation, pegylation, or any similar process whichretains the biological or immunological function of the polypeptide fromwhich it was derived.

[0078] The term “homology”, as used herein, refers to a degree ofcomplementarity. There may be partial homology or complete homology(i.e., identity). A partially complementary sequence that at leastpartially inhibits an identical sequence from hybridizing to a targetnucleic acid is referred to using the functional term “substantiallyhomologous.” The inhibition of hybridization of the completelycomplementary sequence to the target sequence may be examined using ahybridization assay (Southern or northern blot, solution hybridizationand the like) under conditions of low stringency. A substantiallyhomologous sequence or hybridization probe will compete for and inhibitthe binding of a completely homologous sequence to the target sequenceunder conditions of low stringency. This is not to say that conditionsof low stringency are such that non-specific binding is permitted; lowstringency conditions require that the binding of two sequences to oneanother be a specific (i.e., selective) interaction. The absence ofnon-specific binding may be tested by the use of a second targetsequence which lacks even a partial degree of complementarity (e.g.,less than about 30% identity). In the absence of non-specific binding,the probe will not hybridize to the second non-complementary targetsequence.

[0079] Human artificial chromosomes (HACs) are linear microchromosomeswhich may contain DNA sequences of 10K to 10M in size and contain all ofthe elements required for stable mitotic chromosome segregation andmaintenance (Harrington, J. J. et al. (1997) Nat Genet. 15:345-355).

[0080] The term “humanized antibody”, as used herein, refers to antibodymolecules in which amino acids have been replaced in the non-antigenbinding regions in order to more closely resemble a human antibody,while still retaining the original binding ability.

[0081] The term “hybridization”, as used herein, refers to any processby which a strand of nucleic acid binds with a complementary strandthrough base pairing.

[0082] The term “hybridization complex”, as used herein, refers to acomplex formed between two nucleic acid sequences by virtue of theformation of hydrogen bonds between complementary G and C bases andbetween complementary A and T bases; these hydrogen bonds may be furtherstabilized by base stacking interactions. The two complementary nucleicacid sequences hydrogen bond in an antiparallel configuration. Ahybridization complex may be formed in solution (e.g., C₀t or R₀tanalysis) or between one nucleic acid sequence present in solution andanother nucleic acid sequence immobilized on a solid support (e.g.,paper, membranes, filters, chips, pins or glass slides, or any otherappropriate substrate to which cells or their nucleic acids have beenfixed).

[0083] An “insertion” or “addition”, as used herein, refers to a changein an amino acid or nucleotide sequence resulting in the addition of oneor more amino acid residues or nucleotides, respectively, as compared tothe naturally occurring molecule.

[0084] “Microarray” refers to an array of distinct polynucleotides oroligonucleotides synthesized on a substrate, such as paper, nylon orother type of membrane, filter, chip, glass slide, or any other suitablesolid support.

[0085] The term “modulate”, as used herein, refers to a change in theactivity of RABP. For example, modulation may cause an increase or adecrease in protein activity, binding characteristics, or any otherbiological, functional or immunological properties of RABP.

[0086] “Nucleic acid sequence”, as used herein, refers to anoligonucleotide, nucleotide, or polynucleotide, and fragments thereof,and to DNA or RNA of genomic or synthetic origin which may be single- ordouble-stranded, and represent the sense or antisense strand.“Fragments” are those nucleic acid sequences which are greater than 60nucleotides than in length, and most preferably includes fragments thatare at least 100 nucleotides or at least 1000 nucleotides, and at least10,000 nucleotides in length.

[0087] The term “oligonucleotide” refers to a nucleic acid sequence ofat least about 6 nucleotides to about 60 nucleotides, preferably about15 to 30 nucleotides, and more preferably about 20 to 25 nucleotides,which can be used in PCR amplification or a hybridization assay, or amicroarray. As used herein, oligonucleotide is substantially equivalentto the terms “amplimers”, “primers”, “oligomers”, and “probes”, ascommonly defined in the art.

[0088] “Peptide nucleic acid”, PNA, as used herein, refers to anantisense molecule or anti-gene agent which comprises an oligonucleotideof at least five nucleotides in length linked to a peptide backbone ofamino acid residues which ends in lysine. The terminal lysine conferssolubility to the composition. PNAs may be pegylated to extend theirlifespan in the cell where they preferentially bind complementary singlestranded DNA and RNA and stop transcript elongation (Nielsen, P. E. etal. (1993) Anticancer Drug Des. 8:53-63).

[0089] The term “portion”, as used herein, with regard to a protein (asin “a portion of a given protein”) refers to fragments of that protein.The fragments may range in size from five amino acid residues to theentire amino acid sequence minus one amino acid. Thus, a protein“comprising at least a portion of the amino acid sequence of SEQ IDNO:1, SEQ ID NO:3, or SEQ ID NO:5” encompasses the full-length RABP andfragments thereof.

[0090] The term “sample”, as used herein, is used in its broadest sense.A biological sample suspected of containing nucleic acid encoding RABP,or fragments thereof, or RABP itself may comprise a bodily fluid,extract from a cell, chromosome, organelle, or membrane isolated from acell, a cell, genomic DNA, RNA, or cDNA (in solution or bound to a solidsupport), a tissue, a tissue print, and the like.

[0091] The terms “specific binding” or “specifically binding”, as usedherein, refer to that interaction between a protein or peptide and anagonist, an antibody and an antagonist. The interaction is dependentupon the presence of a particular structure (i.e., the antigenicdeterminant or epitope) of the protein recognized by the bindingmolecule. For example, if an antibody is specific for epitope “A”, thepresence of a protein containing epitope A (or free, unlabeled A) in areaction containing labeled “A” and the antibody will reduce the amountof labeled A bound to the antibody.

[0092] The terms “stringent conditions” or “stringency”, as used herein,refer to the conditions for hybridization as defined by the nucleicacid, salt, and temperature. These conditions are well known in the artand may be altered in order to identify or detect identical or relatedpolynucleotide sequences. Numerous equivalent conditions comprisingeither low or high stringency depend on factors such as the length andnature of the sequence (DNA, RNA, base composition), nature of thetarget (DNA, RNA, base composition), milieu (in solution or immobilizedon a solid substrate), concentration of salts and other components(e.g., formamide, dextran sulfate and/or polyethylene glycol), andtemperature of the reactions (within a range from about 5° C. below themelting temperature of the probe to about 20° C. to 25° C. below themelting temperature). One or more factors be may be varied to generateconditions of either low or high stringency different from, butequivalent to, the above listed conditions.

[0093] The term “substantially purified”, as used herein, refers tonucleic or amino acid sequences that are removed from their naturalenvironment, isolated or separated, and are at least 60% free,preferably 75% free, and most preferably 90% free from other componentswith which they are naturally associated.

[0094] A “substitution”, as used herein, refers to the replacement ofone or more amino acids or nucleotides by different amino acids ornucleotides, respectively.

[0095] “Transformation”, as defined herein, describes a process by whichexogenous DNA enters and changes a recipient cell. It may occur undernatural or artificial conditions using various methods well known in theart. Transformation may rely on any known method for the insertion offoreign nucleic acid sequences into a prokaryotic or eukaryotic hostcell. The method is selected based on the type of host cell beingtransformed and may include, but is not limited to, viral infection,electroporation, heat shock, lipofection, and particle bombardment. Such“transformed” cells include stably transformed cells in which theinserted DNA is capable of replication either as an autonomouslyreplicating plasmid or as part of the host chromosome. They also includecells which transiently express the inserted DNA or RNA for limitedperiods of time.

[0096] A “variant” of RABP, as used herein, refers to an amino acidsequence that is altered by one or more amino acids. The variant mayhave “conservative” changes, wherein a substituted amino acid hassimilar structural or chemical properties, e.g., replacement of leucinewith isoleucine. More rarely, a variant may have “nonconservative”changes, e.g., replacement of a glycine with a tryptophan. Analogousminor variations may also include amino acid deletions or insertions, orboth. Guidance in determining which amino acid residues may besubstituted, inserted, or deleted without abolishing biological orimmunological activity may be found using computer programs well knownin the art, for example, DNASTAR software.

[0097] The Invention

[0098] The invention is based on the discovery of three new human Rabproteins (hereinafter collectively referred to as “RABP”), thepolynucleotides encoding RABP, and the use of these compositions for thediagnosis, prevention, or treatment of inflammation and disordersassociated with cell proliferation and apoptosis.

[0099] Nucleic acids encoding the RABP-1 of the present invention werefirst identified in Incyte Clone 2312652 from a tumorous neurogangliontissue cDNA library (NGANNOT01) using a computer search for amino acidsequence alignments. A consensus sequence, SEQ ID NO:2, was derived fromthe following overlapping and/or extended nucleic acid sequences: IncyteClones 2312652 (NGANNOT01), 1851992 (LUNGFET03), 1234510 (LUNGFET03),1880394 (LEUKNOT03), 2209748 (SINTFET03), 1439042 (PANCNOT08), and487135 (HNT2AGT01).

[0100] Nucleic acids encoding the RABP-2 of the present invention werefirst identified in Incyte Clone 2514506 from a liver tumor tissue cDNAlibrary (LIVRTUT04) using a computer search for amino acid sequencealignments. A consensus sequence, SEQ ID NO:4, was derived from thefollowing overlapping and/or extended nucleic acid sequences: IncyteClones 2530505 (GBLANOT02), 1400140 (BRAITUT08), 2619847 (KERANOT02),and 2514506 (LIVRTUT04).

[0101] Nucleic acids encoding the RABP-3 of the present invention werefirst identified in Incyte Clone 3400003 from a nontumorous corpuscavemosum tissue cDNA library (UTRSNOT16) using a computer search foramino acid sequence alignments. A consensus sequence, SEQ ID NO:6, wasderived from the following overlapping and/or extended nucleic acidsequences: Incyte Clones 3400003 (UTRSNOT16), 3440023 (PENCNOT06),1804977 (SINTNOT13), 938735 (CERVNOT01), 473298 (MMLR1DT01), 882035(THYRNOT02), and 623472 (PGANNOT01).

[0102] In one embodiment, the invention encompasses a polypeptide,RABP-1, comprising the amino acid sequence of SEQ ID NO:1, as shown inFIGS. 1A, 1B, 1C, and 1D. RABP-1 is 203 amino acids in length. It hasfour conserved GTP-binding sites encompassing residues G14-K20, D63-E68,T120-D123, and E152-K156, analogous to other LMW GTP-binding proteins.It also has the conserved arginine, R79, and phenylalanine, F165. Theeffector site for binding GAP encompasses residues T40-F45. The carboxyterminus of RABP-1 has the two conserved cysteine residues for bindinglipid. RABP-1 has six potential casein kinase II phosphorylation sitesencompassing residues S22-E25, S51-D54, S94-E97, S108-E111, T120-D123,and S160-E163, and two potential protein kinase C phosphorylation sitesencompassing residues S67-R69 and S154-K156. As shown in FIG. 4, RABP-1has chemical and structural homology with a mouse Rab24 (GI 438164; SEQID NO:7). In particular, RABP and the mouse Rab24 share 97% sequencehomology. As illustrated by FIGS. 7A and 7B, RABP-1 and the mouse Rab24have rather similar hydrophobicity plots. Northern analysis shows theexpression of RABP-1 in various cDNA libraries, at least 29% of whichare immortalized or cancerous, at least 19% of which involve immuneresponse, and at least 26% are expressed in fetal/infant tissues ororgans.

[0103] In another embodiment, the invention encompasses a polypeptide,RABP-2, comprising the amino acid sequence of SEQ ID NO:3, as shown inFIGS. 2A, 2B, and 2C. RABP-2 is 201 amino acids in length. It has fourconserved GTP-binding sites encompassing residues G15-S22, D63-E68,N121-D124, and E149-K153, analogous to other LMW GTP-binding proteins.It also has the conserved arginine, R79, and phenylalanine, F162. Theeffector site for binding GAP encompasses residues T40-F45. The carboxyterminus of RABP-2 has the two conserved cysteine residues for bindinglipid. RABP-2 has three potential N-glycosylation sites encompassingresidues N121-D124, N133-A136, and N154-N157; five potential caseinkinase H phosphorylation sites encompassing residues T32-E35, T91-E94,T135-E138, T156-E159, and S179-E182; one potential tyrosine kinasephosphrylation site encompassing residues R27-Y33; and five potentialprotein kinase C phosphorylation sites encompassing residues T56-K58,T126-K128, T127-K129, T135-K137, and S151-K153. As shown in FIG. 5,RABP-2 has chemical and structural homology with a rat Rab1B (GI 57006;SEQ ID NO:8). In particular, RABP and the rat Rab1B share 98% sequencehomology. As illustrated by FIGS. 8A and 8B, RABP-2 and the rat Rab1Bhave rather similar hydrophobicity plots. Northern analysis shows theexpression of RABP-2 in various cDNA libraries, at least 50% of whichare immortalized or cancerous, at least 14% of which involve immuneresponse, and at least 14% are expressed in fetal/infant tissues ororgans.

[0104] In a further embodiment, the invention encompasses a polypeptide,RABP-3, comprising the amino acid sequence of SEQ ID NO:5, as shown inFIGS. 3A, 3B, 3C, 3D, 3E, 3F, 3G, and 3H. RABP-3 is 221 amino acids inlength. It has four conserved GTP-binding sites encompassing residuesG19-K25, D68-T73, N129-D132, and F157-K161, analogous to other LMWGTP-binding proteins. The effector site for binding GAP encompassesresidues T44-F49. The carboxy terminus of RABP-3 has a conserved CAAXbox encompassing residues C218-G221 for binding lipid. RABP-3 has onepotential N-glycosylation site encompassing residues N61-L64; twopotential cAMP- and cGMP-dependent protein kinase phosphorylation sitesencompassing residues R52-T55 and K112-S115; one potential casein kinaseII phosphorylation sites encompassing residues S2-E5 and S4-E7; onepotential tyrosine kinase phosphrylation site encompassing residuesK77-Y84; and four potential protein kinase C phosphorylation sitesencompassing residues S23-K25, T138-K140, S159-K161, and S190-R192. Asshown in FIG. 6, RABP-3 has chemical and structural homology with a ratRab28 (GI 1154901; SEQ ID NO:9). In particular, RABP and the rat Rab28share 92% sequence homology. As illustrated by FIGS. 9A and 9B, RABP-3and the rat Rab28 have rather similar hydrophobicity plots. Northernanalysis shows the expression of RABP-3 in various cDNA libraries, atleast 50% of which are immortalized or cancerous, at least 21% of whichinvolve immune response, and at least 11% are expressed in fetal/infanttissues or organs.

[0105] The invention also encompasses RABP variants. A preferred RABPvariant is one having at least 80%, and more preferably at least 90%,amino acid sequence identity to the RABP amino acid sequence (SEQ IDNO:1, SEQ ID NO:3, or SEQ ID NO:5) and which retains at least onebiological, immunological or other functional characteristic or activityof RABP. A most preferred RABP variant is one having at least 95% aminoacid sequence identity to SEQ ID NO:1, SEQ ID NO:3, or SEQ ID NO:5.

[0106] The invention also encompasses polynucleotides which encode RABP.Accordingly, any nucleic acid sequence which encodes the amino acidsequence of RABP can be used to produce recombinant molecules whichexpress RABP. In a particular embodiment, the invention encompasses thepolynucleotide comprising the nucleic acid sequence of SEQ ID NO:2, SEQID NO:4, or SEQ ID NO:6 as shown in FIGS. 1A, 1B, 1C, and 1D, FIGS. 2A,2B, and 2C, or FIGS. 3A, 3B, 3C, 3D, 3E, 3F, 3G, and 3H.

[0107] It will be appreciated by those skilled in the art that as aresult of the degeneracy of the genetic code, a multitude of nucleotidesequences encoding RABP, some bearing minimal homology to the nucleotidesequences of any known and naturally occurring gene, may be produced.Thus, the invention contemplates each and every possible variation ofnucleotide sequence that could be made by selecting combinations basedon possible codon choices. These combinations are made in accordancewith the standard triplet genetic code as applied to the nucleotidesequence of naturally occurring RABP, and all such variations are to beconsidered as being specifically disclosed.

[0108] Although nucleotide sequences which encode RABP and its variantsare preferably capable of hybridizing to the nucleotide sequence of thenaturally occurring RABP under appropriately selected conditions ofstringency, it may be advantageous to produce nucleotide sequencesencoding RABP or its derivatives possessing a substantially differentcodon usage. Codons may be selected to increase the rate at whichexpression of the peptide occurs in a particular prokaryotic oreukaryotic host in accordance with the frequency with which particularcodons are utilized by the host. Other reasons for substantiallyaltering the nucleotide sequence encoding RABP and its derivativeswithout altering the encoded amino acid sequences include the productionof RNA transcripts having more desirable properties, such as a greaterhalf-life, than transcripts produced from the naturally occurringsequence.

[0109] The invention also encompasses production of DNA sequences, orfragments thereof, which encode RABP and its derivatives, entirely bysynthetic chemistry. After production, the synthetic sequence may beinserted into any of the many available expression vectors and cellsystems using reagents that are well known in the art. Moreover,synthetic chemistry may be used to introduce mutations into a sequenceencoding RABP or any fragment thereof.

[0110] Also encompassed by the invention are polynucleotide sequencesthat are capable of hybridizing to the claimed nucleotide sequences, andin particular, those shown in SEQ ID NO:2, SEQ ID NO:4, or SEQ ID NO:6,under various conditions of stringency as taught in Wahl, G. M. and S.L. Berger (1987; Methods Enzymol. 152:399-407) and Kimmel, A. R. (1987;Methods Enzymol. 152:507-511).

[0111] Methods for DNA sequencing which are well known and generallyavailable in the art and may be used to practice any of the embodimentsof the invention. The methods may employ such enzymes as the Klenowfragment of DNA polymerase I, SEQUENASE (US Biochemical Corp, Cleveland,Ohio), Taq polymerase (Perkin Elmer), thermostable T7 polymerase(Amersham, Chicago, Ill.), or combinations of polymerases andproofreading exonucleases such as those found in the ELONGASEAmplification System marketed by Gibco/BRL (Gaithersburg, Md.).Preferably, the process is automated with machines such as the HamiltonMicro Lab 2200 (Hamilton, Reno, Nev.), Peltier Thermal Cycler (PTC200;MJ Research, Watertown, Mass.) and the ABI Catalyst and 373 and 377 DNASequencers (Perkin Elmer).

[0112] The nucleic acid sequences encoding RABP may be extendedutilizing a partial nucleotide sequence and employing various methodsknown in the art to detect upstream sequences such as promoters andregulatory elements. For example, one method which may be employed,“restriction-site” PCR, uses universal primers to retrieve unknownsequence adjacent to a known locus (Sarkar, G. (1993) PCR MethodsApplic. 2:318-322). In particular, genomic DNA is first amplified in thepresence of primer to a linker sequence and a primer specific to theknown region. The amplified sequences are then subjected to a secondround of PCR with the same linker primer and another specific primerinternal to the first one. Products of each round of PCR are transcribedwith an appropriate RNA polymerase and sequenced using reversetranscriptase.

[0113] Inverse PCR may also be used to amplify or extend sequences usingdivergent primers based on a known region (Triglia, T. et al. (1988)Nucleic Acids Res. 16:8186). The primers may be designed usingcommercially available software such as OLIGO 4.06 Primer Analysissoftware (National Biosciences Inc., Plymouth, Minn.), or anotherappropriate program, to be 22-30 nucleotides in length, to have a GCcontent of 50% or more, and to anneal to the target sequence attemperatures about 68°-72° C. The method uses several restrictionenzymes to generate a suitable fragment in the known region of a gene.The fragment is then circularized by intramolecular ligation and used asa PCR template.

[0114] Another method which may be used is capture PCR which involvesPCR amplification of DNA fragments adjacent to a known sequence in humanand yeast artificial chromosome DNA (Lagerstrom, M. et al. (1991) PCRMethods Applic. 1:111-119). In this method, multiple restriction enzymedigestions and ligations may also be used to place an engineereddouble-stranded sequence into an unknown fragment of the DNA moleculebefore performing PCR.

[0115] Another method which may be used to retrieve unknown sequences isthat of Parker, J. D. et al. (1991; Nucleic Acids Res. 19:3055-3060).Additionally, one may use PCR, nested primers, and PROMOTERFINDERlibraries to walk genomic DNA (Clontech, Palo Alto, Calif.). Thisprocess avoids the need to screen libraries and is useful in findingintron/exon junctions.

[0116] When screening for full-length cDNAs, it is preferable to uselibraries that have been size-selected to include larger cDNAs. Also,random-primed libraries are preferable, in that they will contain moresequences which contain the 5′ regions of genes. Use of a randomlyprimed library may be especially preferable for situations in which anoligo d(T) library does not yield a full-length cDNA. Genomic librariesmay be useful for extension of sequence into 5′ non-transcribedregulatory regions.

[0117] Capillary electrophoresis systems which are commerciallyavailable may be used to analyze the size or confirm the nucleotidesequence of sequencing or PCR products. In particular, capillarysequencing may employ flowable polymers for electrophoretic separation,four different fluorescent dyes (one for each nucleotide) which arelaser activated, and detection of the emitted wavelengths by a chargecoupled device camera. Output/light intensity may be converted toelectrical signal using appropriate software (e.g. GENOTYPER andSEQUENCE NAVIGATOR, Perkin Elmer) and the entire process from loading ofsamples to computer analysis and electronic data display may be computercontrolled. Capillary electrophoresis is especially preferable for thesequencing of small pieces of DNA which might be present in limitedamounts in a particular sample.

[0118] In another embodiment of the invention, polynucleotide sequencesor fragments thereof which encode RABP may be used in recombinant DNAmolecules to direct expression of RABP, fragments or functionalequivalents thereof, in appropriate host cells. Due to the inherentdegeneracy of the genetic code, other DNA sequences which encodesubstantially the same or a functionally equivalent amino acid sequencemay be produced, and these sequences may be used to clone and expressRABP.

[0119] As will be understood by those of skill in the art, it may beadvantageous to produce RABP-encoding nucleotide sequences possessingnon-naturally occurring codons. For example, codons preferred by aparticular prokaryotic or eukaryotic host can be selected to increasethe rate of protein expression or to produce an RNA transcript havingdesirable properties, such as a half-life which is longer than that of atranscript generated from the naturally occurring sequence.

[0120] The nucleotide sequences of the present invention can beengineered using methods generally known in the art in order to alterRABP encoding sequences for a variety of reasons, including but notlimited to, alterations which modify the cloning, processing, and/orexpression of the gene product. DNA shuffling by random fragmentationand PCR reassembly of gene fragments and synthetic oligonucleotides maybe used to engineer the nucleotide sequences. For example, site-directedmutagenesis may be used to insert new restriction sites, alterglycosylation patterns, change codon preference, produce splicevariants, introduce mutations, and so forth.

[0121] In another embodiment of the invention, natural, modified, orrecombinant nucleic acid sequences encoding RABP may be ligated to aheterologous sequence to encode a fusion protein. For example, to screenpeptide libraries for inhibitors of RABP activity, it may be useful toencode a chimeric RABP protein that can be recognized by a commerciallyavailable antibody. A fusion protein may also be engineered to contain acleavage site located between the RABP encoding sequence and theheterologous protein sequence, so that RABP may be cleaved and purifiedaway from the heterologous moiety.

[0122] In another embodiment, sequences encoding RABP may besynthesized, in whole or in part, using chemical methods well known inthe art (see Caruthers, M. H. et al. (1980) Nucl. Acids Res. Symp. Ser.215-223, Horn, T. et al. (1980) Nucl. Acids Res. Symp. Ser. 225-232).Alternatively, the protein itself may be produced using chemical methodsto synthesize the amino acid sequence of RABP, or a fragment thereof.For example, peptide synthesis can be performed using varioussolid-phase techniques (Roberge, J. Y. et al. (1995) Science269:202-204) and automated synthesis may be achieved, for example, usingthe ABI 431A Peptide Synthesizer (Perkin Elmer).

[0123] The newly synthesized peptide may be substantially purified bypreparative high performance liquid chromatography (e.g., Creighton, T.(1983) Proteins, Structures and Molecular Principles, WH Freeman andCo., New York, N.Y.). The composition of the synthetic peptides may beconfirmed by amino acid analysis or sequencing (e.g., the Edmandegradation procedure; Creighton, supra). Additionally, the amino acidsequence of RABP, or any part thereof, may be altered during directsynthesis and/or combined using chemical methods with sequences fromother proteins, or any part thereof, to produce a variant polypeptide.

[0124] In order to express a biologically active RABP, the nucleotidesequences encoding RABP or functional equivalents, may be inserted intoappropriate expression vector, i.e., a vector which contains thenecessary elements for the transcription and translation of the insertedcoding sequence.

[0125] Methods which are well known to those skilled in the art may beused to construct expression vectors containing sequences encoding RABPand appropriate transcriptional and translational control elements.These methods include in vitro recombinant DNA techniques, synthetictechniques, and in vivo genetic recombination. Such techniques aredescribed in Sambrook, J. et al. (1989) Molecular Cloning, A LaboratoryManual, Cold Spring Harbor Press, Plainview, N.Y., and Ausubel, F. M. etal. (1989) Current Protocols in Molecular Biology, John Wiley & Sons,New York, N.Y.

[0126] A variety of expression vector/host systems may be utilized tocontain and express sequences encoding RABP. These include, but are notlimited to, microorganisms such as bacteria transformed with recombinantbacteriophage, plasmid, or cosmid DNA expression vectors; yeasttransformed with yeast expression vectors; insect cell systems infectedwith virus expression vectors (e.g., baculovirus); plant cell systemstransformed with virus expression vectors (e.g., cauliflower mosaicvirus, CaMV; tobacco mosaic virus, TMV) or with bacterial expressionvectors (e.g., Ti or pBR322 plasmids); or animal cell systems. Theinvention is not limited by the host cell employed.

[0127] The “control elements” or “regulatory sequences” are thosenon-translated regions of the vector—enhancers, promoters, 5′ and 3′untranslated regions—which interact with host cellular proteins to carryout transcription and translation. Such elements may vary in theirstrength and specificity. Depending on the vector system and hostutilized, any number of suitable transcription and translation elements,including constitutive and inducible promoters, may be used. Forexample, when cloning in bacterial systems, inducible promoters such asthe hybrid lacZ promoter of the BLUESCRIPT phagemid (Stratagene,LaJolla, Calif.) or PSPORT1 plasmid (Gibco BRL) and the like may beused. The baculovirus polyhedrin promoter may be used in insect cells.Promoters or enhancers derived from the genomes of plant cells (e.g.,heat shock, RUBISCO; and storage protein genes) or from plant viruses(e.g., viral promoters or leader sequences) may be cloned into thevector. In mammalian cell systems, promoters from mammalian genes orfrom mammalian viruses are preferable. If it is necessary to generate acell line that contains multiple copies of the sequence encoding RABP,vectors based on SV40 or EBV may be used with an appropriate selectablemarker.

[0128] In bacterial systems, a number of expression vectors may beselected depending upon the use intended for RABP. For example, whenlarge quantities of RABP are needed for the induction of antibodies,vectors which direct high level expression of fusion proteins that arereadily purified may be used. Such vectors include, but are not limitedto, the multifunctional E. coli cloning and expression vectors such asBLUESCRIPT (Stratagene), in which the sequence encoding RABP may beligated into the vector in frame with sequences for the amino-terminalMet and the subsequent 7 residues of β-galactosidase so that a hybridprotein is produced; pIN vectors (Van Heeke, G. and S. M. Schuster(1989) J. Biol. Chem. 264:5503-5509); and the like. pGEX vectors(Promega, Madison, Wis.) may also be used to express foreignpolypeptides as fusion proteins with glutathione S-transferase (GST). Ingeneral, such fusion proteins are soluble and can easily be purifiedfrom lysed cells by adsorption to glutathione-agarose beads followed byelution in the presence of free glutathione. Proteins made in suchsystems may be designed to include heparin, thrombin, or factor XAprotease cleavage sites so that the cloned polypeptide of interest canbe released from the GST moiety at will.

[0129] In the yeast, Saccharomyces cerevisiae, a number of vectorscontaining constitutive or inducible promoters such as alpha factor,alcohol oxidase, and PGH may be used. For reviews, see Ausubel et al.(supra) and Grant et al. (1987) Methods Enzymol. 153:516-544.

[0130] In cases where plant expression vectors are used, the expressionof sequences encoding RABP may be driven by any of a number ofpromoters. For example, viral promoters such as the 35S and 19Spromoters of CaMV may be used alone or in combination with the omegaleader sequence from TMV (Takamatsu, N. (1987) EMBO J. 6:307-311).Alternatively, plant promoters such as the small subunit of RUBISCO orheat shock promoters may be used (Coruzzi, G. et al. (1984) EMBO J.3:1671-1680; Broglie, R. et al. (1984) Science 224:838-843; and Winter,J. et al. (1991) Results Probl. Cell Differ. 17:85-105). Theseconstructs can be introduced into plant cells by direct DNAtransformation or pathogen-mediated transfection. Such techniques aredescribed in a number of generally available reviews (see, for example,Hobbs, S. or Murry, L. E. in McGraw Hill Yearbook of Science andTechnology (1992) McGraw Hill, New York, N.Y.; pp. 191-196).

[0131] An insect system may also be used to express RABP. For example,in one such system, Autographa californica nuclear polyhedrosis virus(AcNPV) is used as a vector to express foreign genes in Spodopterafrugiperda cells or in Trichoplusia larvae. The sequences encoding RABPmay be cloned into a non-essential region of the virus, such as thepolyhedrin gene, and placed under control of the polyhedrin promoter.Successful insertion of RABP will render the polyhedrin gene inactiveand produce recombinant virus lacking coat protein. The recombinantviruses may then be used to infect, for example, S. frugiperda cells orTrichoplusia larvae in which RABP may be expressed (Engelhard, E. K. etal. (1994) Proc. Nat. Acad. Sci. 91:3224-3227).

[0132] In mammalian host cells, a number of viral-based expressionsystems may be utilized. In cases where an adenovirus is used as anexpression vector, sequences encoding RABP may be ligated into anadenovirus transcription/translation complex consisting of the latepromoter and tripartite leader sequence. Insertion in a non-essential E1or E3 region of the viral genome may be used to obtain a viable viruswhich is capable of expressing RABP in infected host cells (Logan, J.and Shenk, T. (1984) Proc. Natl. Acad. Sci. 81:3655-3659). In addition,transcription enhancers, such as the Rous sarcoma virus (RSV) enhancer,may be used to increase expression in mammalian host cells.

[0133] Human artificial chromosomes (HACs) may also be employed todeliver larger fragments of DNA than can be contained and expressed in aplasmid. HACs of 6 to 10M are constructed and delivered via conventionaldelivery methods (lippsomes, polycationic amino polymers, or vesicles)for therapeutic purposes.

[0134] Specific initiation signals may also be used to achieve moreefficient translation of sequences encoding RABP. Such signals includethe ATG initiation codon and adjacent sequences. In cases wheresequences encoding RABP, its initiation codon, and upstream sequencesare inserted into the appropriate expression vector, no additionaltranscriptional or translational control signals may be needed. However,in cases where only coding sequence, or a fragment thereof, is inserted,exogenous translational control signals including the ATG initiationcodon should be provided. Furthermore, the initiation codon should be inthe correct reading frame to ensure translation of the entire insert.Exogenous translational elements and initiation codons may be of variousorigins, both natural and synthetic. The efficiency of expression may beenhanced by the inclusion of enhancers which are appropriate for theparticular cell system which is used, such as those described in theliterature (Scharf, D. et al. (1994) Results Probl. Cell Differ.20:125-162).

[0135] In addition, a host cell strain may be chosen for its ability tomodulate the expression of the inserted sequences or to process theexpressed protein in the desired fashion. Such modifications of thepolypeptide include, but are not limited to, acetylation, carboxylation,glycosylation, phosphorylation, lipidation, and acylation.Post-translational processing which cleaves a “prepro” form of theprotein may also be used to facilitate correct insertion, folding and/orfunction. Different host cells which have specific cellular machineryand characteristic mechanisms for post-translational activities (e.g.,CHO, HeLa, MDCK, HEK293, and WI38), are available from the American TypeCulture Collection (ATCC; Bethesda, Md.) and may be chosen to ensure thecorrect modification and processing of the foreign protein.

[0136] For long-term, high-yield production of recombinant proteins,stable expression is preferred. For example, cell lines which stablyexpress RABP may be transformed using expression vectors which maycontain viral origins of replication and/or endogenous expressionelements and a selectable marker gene on the same or on a separatevector. Following the introduction of the vector, cells may be allowedto grow for 1-2 days in an enriched media before they are switched toselective media. The purpose of the selectable marker is to conferresistance to selection, and its presence allows growth and recovery ofcells which successfully express the introduced sequences. Resistantclones of stably transformed cells may be proliferated using tissueculture techniques appropriate to the cell type.

[0137] Any number of selection systems may be used to recovertransformed cell lines. These include, but are not limited to, theherpes simplex virus thymidine kinase (Wigler, M. et al. (1977) Cell11:223-32) and adenine phosphoribosyltransferase (Lowy, I. et al. (1980)Cell 22:817-23) genes which can be employed in tk⁻ or aprt⁻ cells,respectively. Also, antimetabolite, antibiotic or herbicide resistancecan be used as the basis for selection; for example, dhfr which confersresistance to methotrexate (Wigler, M. et al. (1980) Proc. Natl. Acad.Sci. 77:3567-70); npt, which confers resistance to the aminoglycosides,neomycin and G-418 (Colbere-Garapin, F. et al (1981) J. Mol. Biol.150:1-14); and als or pat, which confer resistance to chlorsulfuron andphosphinotricin acetyltransferase, respectively (Murry, supra).Additional selectable genes have been described, for example, trpB,which allows cells to utilize indole in place of tryptophan, or hisD,which allows cells to utilize histinol in place of histidine (Hartman,S. C. and R. C. Mulligan (1988) Proc. Natl. Acad. Sci. 85:8047-51).Recently, the use of visible markers has gained popularity with suchmarkers as anthocyanins, β glucuronidase and its substrate GUS, andluciferase and its substrate luciferin, being widely used not only toidentify transformants, but also to quantify the amount of transient orstable protein expression attributable to a specific vector system(Rhodes, C. A. et al. (1995) Methods Mol. Biol. 55:121-131).

[0138] Although the presence/absence of marker gene expression suggeststhat the gene of interest is also present, its presence and expressionmay need to be confirmed. For example, if the sequence encoding RABP isinserted within a marker gene sequence, transformed cells containingsequences encoding RABP can be identified by the absence of marker genefunction. Alternatively, a marker gene can be placed in tandem with asequence encoding RABP under the control of a single promoter.Expression of the marker gene in response to induction or selectionusually indicates expression of the tandem gene as well.

[0139] Alternatively, host cells which contain the nucleic acid sequenceencoding RABP and express RABP may be identified by a variety ofprocedures known to those of skill in the art. These procedures include,but are not limited to, DNA-DNA or DNA-RNA hybridizations and proteinbioassay or immunoassay techniques which include membrane, solution, orchip based technologies for the detection and/or quantification ofnucleic acid or protein.

[0140] The presence of polynucleotide sequences encoding RABP can bedetected by DNA-DNA or DNA-RNA hybridization or amplification usingprobes or fragments or fragments of polynucleotides encoding RABP.Nucleic acid amplification based assays involve the use ofoligonucleotides or oligomers based on the sequences encoding RABP todetect transformants containing DNA or RNA encoding RABP.

[0141] A variety of protocols for detecting and measuring the expressionof RABP, using either polyclonal or monoclonal antibodies specific forthe protein are known in the art. Examples include enzyme-linkedimmunosorbent assay (ELISA), radioimmunoassay (RIA), and fluorescenceactivated cell sorting (FACS). A two-site, monoclonal-based immunoassayutilizing monoclonal antibodies reactive to two non-interfering epitopeson RABP is preferred, but a competitive binding assay may be employed.These and other assays are described, among other places, in Hampton, R.et al. (1990; Serological Methods, a Laboratory Manual, APS Press, StPaul, Minn.) and Maddox, D. E. et al. (1983; J. Exp. Med.158:1211-1216).

[0142] A wide variety of labels and conjugation techniques are known bythose skilled in the art and may be used in various nucleic acid andamino acid assays. Means for producing labeled hybridization or PCRprobes for detecting sequences related to polynucleotides encoding RABPinclude oligolabeling, nick translation, end-labeling or PCRamplification using a labeled nucleotide. Alternatively, the sequencesencoding RABP, or any fragments thereof may be cloned into a vector forthe production of an mRNA probe. Such vectors are known in the art, arecommercially available, and may be used to synthesize RNA probes invitro by addition of an appropriate RNA polymerase such as T7, T3, orSP6 and labeled nucleotides. These procedures may be conducted using avariety of commercially available kits (Pharmacia & Upjohn, (Kalamazoo,Mich.); Promega (Madison Wis.); and U.S. Biochemical Corp., (Cleveland,Ohio)). Suitable reporter molecules or labels, which may be used forease of detection, include radionuclides, enzymes, fluorescent,chemiluminescent, or chromogenic agents as well as substrates,cofactors, inhibitors, magnetic particles, and the like.

[0143] Host cells transformed with nucleotide sequences encoding RABPmay be cultured under conditions suitable for the expression andrecovery of the protein from cell culture. The protein produced by atransformed cell may be secreted or contained intracellularly dependingon the sequence and/or the vector used. As will be understood by thoseof skill in the art, expression vectors containing polynucleotides whichencode RABP may be designed to contain signal sequences which directsecretion of RABP through a prokaryotic or eukaryotic cell membrane.Other constructions may be used to join sequences encoding RABP tonucleotide sequence encoding a polypeptide domain which will facilitatepurification of soluble proteins. Such purification facilitating domainsinclude, but are not limited to, metal chelating peptides such ashistidine-tryptophan modules that allow purification on immobilizedmetals, protein A domains that allow purification on immobilizedimmunoglobulin, and the domain utilized in the FLAGS extension/affinitypurification system (Immunex Corp., Seattle, Wash.). The inclusion ofcleavable linker sequences such as those specific for Factor XA orenterokinase (Invitrogen, San Diego, Calif.) between the purificationdomain and RABP may be used to facilitate purification. One suchexpression vector provides for expression of a fusion protein containingRABP and a nucleic acid encoding 6 histidine residues preceding athioredoxin or an enterokinase cleavage site. The histidine residuesfacilitate purification on IMAC (immobilized metal ion affinitychromatography) as described in Porath, J. et al. (1992, Prot. Exp.Purif. 3: 263-281) while the enterokinase cleavage site provides a meansfor purifying RABP from the fusion protein. A discussion of vectorswhich contain fusion proteins is provided in Kroll, D. J. et al. (1993;DNA Cell Biol. 12:441-453).

[0144] In addition to recombinant production, fragments of RABP may beproduced by direct peptide synthesis using solid-phase techniques(Merrifield J. (1963) J. Am. Chem. Soc. 85:2149-2154). Protein synthesismay be performed using manual techniques or by automation. Automatedsynthesis may be achieved, for example, using Applied Biosystems 431APeptide Synthesizer (Perkin Elmer). Various fragments of RABP may bechemically synthesized separately and combined using chemical methods toproduce the full length molecule.

[0145] Therapeutics

[0146] Chemical and structural homology exists between RABP-1 and amouse Rab24 (GI 438164; SEQ ID NO:7), between RABP-2 and a rat Rab1B (GI57006; SEQ ID NO:8), and between RABP-3 and a rat Rab28 (GI 1154901; SEQID NO:9). Northern analysis shows that the expression of RABP (SEQ IDNO:1, SEQ ID NO:3, or SEQ ID NO:5) is associated with cancer andfetal/infant development. Therapeutic uses for all three polypeptidesare described collectively below.

[0147] During fetal development, decreased expression of RABP may causean increase in apoptosis with no adverse effects to the subject.However, in other situations and in adults, decreased expression of RABPmay cause an increase in apoptosis which is detrimental. Therefore, inone embodiment, RABP or a fragment or derivative thereof may beadministered to a subject to prevent or treat a disorder associated withan increase in apoptosis. Such disorders include, but are not limitedto, AIDS and other infectious or genetic immunodeficiencies,neurodegenerative diseases such as Alzheimer's disease, Parkinson'sdisease, amyotrophic lateral sclerosis, retinitis pigmentosa, andcerebellar degeneration, myelodysplastic syndromes such as aplasticanemia, ischemic injuries such as myocardial infarction, stroke, andreperfusion injury, toxin-induced diseases such as alcohol-induced liverdamage, cirrhosis, and lathyrism, wasting diseases such as cachexia,viral infections such as those caused by hepatitis B and C, andosteoporosis.

[0148] In another embodiment, an agonist which is specific for RABP maybe used to prevent or treat a disorder associated with increasedapoptosis including, but not limited to, those listed above.

[0149] In still another embodiment, a vector capable of expressing RABP(SEQ ID NO:1,), or a fragment or a derivative thereof, may be used toprevent or treat a disorder associated with increased apoptosisincluding, but not limited to, those listed above.

[0150] In a further embodiment, RABP or a fragment or derivative thereofmay be added to cells to stimulate cell proliferation. In particular,RABP may be added to a cell or cells in vivo using delivery mechanismssuch as liposomes, viral based vectors, or electroinjection for thepurpose of promoting regeneration or cell differentiation of the cell orcells. In addition, RABP may be added to a cell, cell line, tissue ororgan culture in vitro or ex vivo to stimulate cell proliferation foruse in heterologous or autologous transplantation. In some cases, thecell will have been selected for its ability to fight an infection or acancer or to correct a genetic defect in a disease such as sickle cellanemia, β thalassemia, cystic fibrosis, or Huntington's chorea.

[0151] In another further embodiment, an agonist which is specific forRABP may be administered to a cell to stimulate cell proliferation, asdescribed above.

[0152] In another further embodiment, a vector capable of expressingRABP, or a fragment or a derivative thereof, may be administered to acell or cells in vivo using delivery mechanisms, or to a cell tostimulate cell proliferation, as described above.

[0153] Increased expression of RABP appears to be associated withincreased cell proliferation. Therefore, in one embodiment, anantagonist of RABP, or a fragment or a derivative thereof, may beadministered to a subject to prevent or treat a disorder associated withcell proliferation. Such disorders include various types of cancerincluding, but not limited to, adenocarcinoma, leukemia, lymphoma,melanoma, myeloma, sarcoma, and teratocarcinoma, and, in particular,cancers of the adrenal gland, bladder, bone, bone marrow, brain, breast,cervix, gall bladder, ganglia, gastrointestinal tract, heart, kidney,liver, lung, muscle, ovary, pancreas, parathyroid, penis, prostate,salivary glands, skin, spleen, testis, thymus, thyroid, and uterus. Inone aspect, an antibody specific for RABP may be used directly as anantagonist, or indirectly as a targeting or delivery mechanism forbringing a pharmaceutical agent to cells or tissue which express RABP.

[0154] In still another embodiment, a vector expressing the complementof the polynucleotide encoding RABP, or a fragment or a derivativethereof, may be administered to a subject to prevent or treat a disorderassociated with cell proliferation including, but not limited to, thetypes of cancer listed above.

[0155] In another embodiment, an antagonist of RABP, or a fragment or aderivative thereof, may be administered to a subject to prevent or treatinflammation. Disorders associated with inflammation include, but arenot limited to, Addison's disease, adult respiratory distress syndrome,allergies, anemia, asthma, atherosclerosis, bronchitis, cholecystitus,Crohn's disease, ulcerative colitis, atopic dermatitis, dermatomyositis,diabetes mellitus, emphysema, atrophic gastritis, glomerulonephritis,gout, Graves' disease, hypereosinophilia, irritable bowel syndrome,lupus erythematosus, multiple sclerosis, myasthenia gravis, myocardialor pericardial inflammation,osteoarthritis, osteoporosis, pancreatitis,polymyositis, rheumatoid arthritis, scleroderma, Sjögren's syndrome, andautoimmune thyroiditis; complications of cancer, hemodialysis,extracorporeal circulation; viral, bacterial, fungal, parasitic,protozoal, and helminthic infections and trauma. In one aspect, anantibody specific for RABP may be used directly as an antagonist, orindirectly as a targeting or delivery mechanism for bringing apharmaceutical agent to cells or tissue which express RABP.

[0156] In still another embodiment, a vector expressing the complementof the polynucleotide encoding RABP, or a fragment or a derivativethereof, may be administered to a subject to prevent or treatinflammation associated with any disorder including, but not limited to,those listed above.

[0157] In other embodiments, any of the proteins, antagonists,antibodies, agonists, complementary sequences or vectors of theinvention may be administered in combination with other appropriatetherapeutic agents. Selection of the appropriate agents for use incombination therapy may be made by one of ordinary skill in the art,according to conventional pharmaceutical principles. The combination oftherapeutic agents may act synergistically to effect the treatment orprevention of the various disorders described above. Using thisapproach, one may be able to achieve therapeutic efficacy with lowerdosages of each agent, thus reducing the potential for adverse sideeffects.

[0158] An antagonist of RABP may be produced using methods which aregenerally known in the art. In particular, purified RABP may be used toproduce antibodies or to screen libraries of pharmaceutical agents toidentify those which specifically bind RABP.

[0159] Antibodies to RABP may be generated using methods that are wellknown in the art. Such antibodies may include, but are not limited to,polyclonal, monoclonal, chimeric, single chain, Fab fragments, andfragments produced by a Fab expression library. Neutralizing antibodies,(i.e., those which inhibit dimer formation) are especially preferred fortherapeutic use.

[0160] For the production of antibodies, various hosts including goats,rabbits, rats, mice, humans, and others, may be immunized by injectionwith RABP or any fragment or oligopeptide thereof which has immunogenicproperties. Depending on the host species, various adjuvants may be usedto increase immunological response. Such adjuvants include, but are notlimited to, Freund's, mineral gels such as aluminum hydroxide, andsurface active substances such as lysolecithin, pluronic polyols,polyanions, peptides, oil emulsions, keyhole limpet hemocyanin, anddinitrophenol. Among adjuvants used in humans, BCG (bacilliCalmette-Guerin) and Corynebacterium parvum are especially preferable.

[0161] It is preferred that the oligopeptides, peptides, or fragmentsused to induce antibodies to RABP have an amino acid sequence consistingof at least five amino acids and more preferably at least 10 aminoacids. It is also preferable that they are identical to a portion of theamino acid sequence of the natural protein, and they may contain theentire amino acid sequence of a small, naturally occurring molecule.Short stretches of RABP amino acids may be fused with those of anotherprotein such as keyhole limpet hemocyanin and antibody produced againstthe chimeric molecule.

[0162] Monoclonal antibodies to RABP may be prepared using any techniquewhich provides for the production of antibody molecules by continuouscell lines in culture. These include, but are not limited to, thehybridoma technique, the human B-cell hybridoma technique, and theEBV-hybridoma technique (Kohler, G. et al. (1975) Nature 256:495-497;Kozbor, D. et al. (1985) J. Immunol. Methods 81:31-42; Cote, R. J. etal. (1983) Proc. Natl. Acad. Sci. 80:2026-2030; Cole, S. P. et al.(1984) Mol. Cell Biol. 62:109-120).

[0163] In addition, techniques developed for the production of “chimericantibodies”, the splicing of mouse antibody genes to human antibodygenes to obtain a molecule with appropriate antigen specificity andbiological activity can be used (Morrison, S. L. et al. (1984) Proc.Natl. Acad. Sci. 81:6851-6855; Neuberger, M. S. et al. (1984) Nature312:604-608; Takeda, S. et al. (1985) Nature 314:452-454).Alternatively, techniques described for the production of single chainantibodies may be adapted, using methods known in the art, to produceRABP-specific single chain antibodies. Antibodies with relatedspecificity, but of distinct idiotypic composition, may be generated bychain shuffling from random combinatorial immunoglobulin libraries(Burton D. R. (1991) Proc. Natl. Acad. Sci. 88:11120-3).

[0164] Antibodies may also be produced by inducing in vivo production inthe lymphocyte population or by screening immunoglobulin libraries orpanels of highly specific binding reagents as disclosed in theliterature (Orlandi, R. et al. (1989) Proc. Natl. Acad. Sci. 86:3833-3837; Winter, G. et al. (1991) Nature 349:293-299).

[0165] Antibody fragments which contain specific binding sites for RABPmay also be generated. For example, such fragments include, but are notlimited to, the F(ab′)2 fragments which can be produced by pepsindigestion of the antibody molecule and the Fab fragments which can begenerated by reducing the disulfide bridges of the F(ab′)2 fragments.Alternatively, Fab expression libraries may be constructed to allowrapid and easy identification of monoclonal Fab fragments with thedesired specificity (Huse, W. D. et al. (1989) Science 254:1275-1281).

[0166] Various immunoassays may be used for screening to identifyantibodies having the desired specificity. Numerous protocols forcompetitive binding or immunoradiometric assays using either polyclonalor monoclonal antibodies with established specificities are well knownin the art. Such immunoassays typically involve the measurement ofcomplex formation between RABP and its specific antibody. A two-site,monoclonal-based immunoassay utilizing monoclonal antibodies reactive totwo non-interfering RABP epitopes is preferred, but a competitivebinding assay may also be employed (Maddox, supra).

[0167] In another embodiment of the invention, the polynucleotidesencoding RABP, or any fragment or complement thereof, may be used fortherapeutic purposes. In one aspect, the complement of thepolynucleotide encoding RABP may be used in situations in which it wouldbe desirable to block the transcription of the mRNA. In particular,cells may be transformed with sequences complementary to polynucleotidesencoding RABP. Thus, complementary molecules or fragments may be used tomodulate RABP activity, or to achieve regulation of gene function. Suchtechnology is now well known in the art, and sense or antisenseoligonucleotides or larger fragments, can be designed from variouslocations along the coding or control regions of sequences encodingRABP.

[0168] Expression vectors derived from retroviruses, adenovirus, herpesor vaccinia viruses, or from various bacterial plasmids may be used fordelivery of nucleotide sequences to the targeted organ, tissue or cellpopulation. Methods which are well known to those skilled in the art canbe used to construct vectors which will express nucleic acid sequencewhich is complementary to the polynucleotides of the gene encoding RABP.These techniques are described both in Sambrook et al. (supra) and inAusubel et al. (supra).

[0169] Genes encoding RABP can be turned off by transforming a cell ortissue with expression vectors which express high levels of apolynucleotide or fragment thereof which encodes RABP. Such constructsmay be used to introduce untranslatable sense or antisense sequencesinto a cell. Even in the absence of integration into the DNA, suchvectors may continue to transcribe RNA molecules until they are disabledby endogenous nucleases. Transient expression may last for a month ormore with a non-replicating vector and even longer if appropriatereplication elements are part of the vector system.

[0170] As mentioned above, modifications of gene expression can beobtained by designing complementary sequences or antisense molecules(DNA, RNA, or PNA) to the control, 5′ or regulatory regions of the geneencoding RABP (signal sequence, promoters, enhancers, and introns).Oligonucleotides derived from the transcription initiation site, e.g.,between positions −10 and +10 from the start site, are preferred.Similarly, inhibition can be achieved using “triple helix” base-pairingmethodology. Triple helix pairing is useful because it causes inhibitionof the ability of the double helix to open sufficiently for the bindingof polymerases, transcription factors, or regulatory molecules. Recenttherapeutic advances using triplex DNA have been described in theliterature (Gee, J. E. et al. (1994) In: Huber, B. E. and B. I. Carr,Molecular and Immunologic Approaches, Futura Publishing Co., Mt. Kisco,N.Y.). The complementary sequence or antisense molecule may also bedesigned to block translation of mRNA by preventing the transcript frombinding to ribosomes.

[0171] Ribozymes, enzymatic RNA molecules, may also be used to catalyzethe specific cleavage of RNA. The mechanism of ribozyme action involvessequence-specific hybridization of the ribozyme molecule tocomplementary target RNA, followed by endonucleolytic cleavage. Exampleswhich may be used include engineered hammerhead motif ribozyme moleculesthat can specifically and efficiently catalyze endonucleolytic cleavageof sequences encoding RABP.

[0172] Specific ribozyme cleavage sites within any potential RNA targetare initially identified by scanning the target molecule for ribozymecleavage sites which include the following sequences: GUA, GUU, and GUC.Once identified, short RNA sequences of between 15 and 20ribonucleotides corresponding to the region of the target genecontaining the cleavage site may be evaluated for secondary structuralfeatures which may render the oligonucleotide inoperable. Thesuitability of candidate targets may also be evaluated by testingaccessibility to hybridization with complementary oligonucleotides usingribonuclease protection assays.

[0173] Complementary ribonucleic acid molecules and ribozymes of theinvention may be prepared by any method known in the art for thesynthesis of nucleic acid molecules. These include techniques forchemically synthesizing oligonucleotides such as solid phasephosphoramidite chemical synthesis. Alternatively, RNA molecules may begenerated by in vitro and in vivo transcription of DNA sequencesencoding RABP. Such DNA sequences may be incorporated into a widevariety of vectors with suitable RNA polymerase promoters such as T7 orSP6. Alternatively, these cDNA constructs that synthesize complementaryRNA constitutively or inducibly can be introduced into cell lines,cells, or tissues.

[0174] RNA molecules may be modified to increase intracellular stabilityand half-life. Possible modifications include, but are not limited to,the addition of flanking sequences at the 5′ and/or 3′ ends of themolecule or the use of phosphorothioate or 2′ O-methyl rather thanphosphodiesterase linkages within the backbone of the molecule. Thisconcept is inherent in the production of PNAs and can be extended in allof these molecules by the inclusion of nontraditional bases such asinosine, queosine, and wybutosine, as well as acetyl-, methyl-, thio-,and similarly modified forms of adenine, cytidine, guanine, thymine, anduridine which are not as easily recognized by endogenous endonucleases.

[0175] Many methods for introducing vectors into cells or tissues areavailable and equally suitable for use in vivo, in vitro, and ex vivo.For ex vivo therapy, vectors may be introduced into stem cells takenfrom the patient and clonally propagated for autologous transplant backinto that same patient. Delivery by transfection, by liposome injectionsor polycationic amino polymers (Goldman, C. K. et al. (1997) NatureBiotechnology 15:462-66; incorporated herein by reference) may beachieved using methods which are well known in the art.

[0176] Any of the therapeutic methods described above may be applied toany subject in need of such therapy, including, for example, mammalssuch as dogs, cats, cows, horses, rabbits, monkeys, and most preferably,humans.

[0177] An additional embodiment of the invention relates to theadministration of a pharmaceutical composition, in conjunction with apharmaceutically acceptable carrier, for any of the therapeutic effectsdiscussed above. Such pharmaceutical compositions may consist of RABP,antibodies to RABP, mimetics, agonists, antagonists, or inhibitors ofRABP. The compositions may be administered alone or in combination withat least one other agent, such as stabilizing compound, which may beadministered in any sterile, biocompatible pharmaceutical carrier,including, but not limited to, saline, buffered saline, dextrose, andwater. The compositions may be administered to a patient alone, or incombination with other agents, drugs or hormones.

[0178] The pharmaceutical compositions utilized in this invention may beadministered by any number of routes including, but not limited to,oral, intravenous, intramuscular, intra-arterial, intramedullary,intrathecal, intraventricular, transdermal, subcutaneous,intraperitoneal, intranasal, enteral, topical, sublingual, or rectalmeans.

[0179] In addition to the active ingredients, these pharmaceuticalcompositions may contain suitable pharmaceutically-acceptable carrierscomprising excipients and auxiliaries which facilitate processing of theactive compounds into preparations which can be used pharmaceutically.Further details on techniques for formulation and administration may befound in the latest edition of Remington's Pharmaceutical Sciences(Maack Publishing Co., Easton, Pa.).

[0180] Pharmaceutical compositions for oral administration can beformulated using pharmaceutically acceptable carriers well known in theart in dosages suitable for oral administration. Such carriers enablethe pharmaceutical compositions to be formulated as tablets, pills,dragees, capsules, liquids, gels, syrups, slurries, suspensions, and thelike, for ingestion by the patient.

[0181] Pharmaceutical preparations for oral use can be obtained throughcombination of active compounds with solid excipient, optionallygrinding a resulting mixture, and processing the mixture of granules,after adding suitable auxiliaries, if desired, to obtain tablets ordragee cores. Suitable excipients are carbohydrate or protein fillers,such as sugars, including lactose, sucrose, mannitol, or sorbitol;starch from corn, wheat, rice, potato, or other plants; cellulose, suchas methyl cellulose, hydroxypropylmethyl-cellulose, or sodiumcarboxymethylcellulose; gums including arabic and tragacanth; andproteins such as gelatin and collagen. If desired, disintegrating orsolubilizing agents may be added, such as the cross-linked polyvinylpyrrolidone, agar, alginic acid, or a salt thereof, such as sodiumalginate.

[0182] Dragee cores may be used in conjunction with suitable coatings,such as concentrated sugar solutions, which may also contain gum arabic,talc, polyvinylpyrrolidone, carbopol gel, polyethylene glycol, and/ortitanium dioxide, lacquer solutions, and suitable organic solvents orsolvent mixtures. Dyestuffs or pigments may be added to the tablets ordragee coatings for product identification or to characterize thequantity of active compound, i.e., dosage.

[0183] Pharmaceutical preparations which can be used orally includepush-fit capsules made of gelatin, as well as soft, sealed capsules madeof gelatin and a coating, such as glycerol or sorbitol. Push-fitcapsules can contain active ingredients mixed with a filler or binders,such as lactose or starches, lubricants, such as talc or magnesiumstearate, and, optionally, stabilizers. In soft capsules, the activecompounds may be dissolved or suspended in suitable liquids, such asfatty oils, liquid, or liquid polyethylene glycol with or withoutstabilizers.

[0184] Pharmaceutical formulations suitable for parenteraladministration may be formulated in aqueous solutions, preferably inphysiologically compatible buffers such as Hanks' solution, Ringer'ssolution, or physiologically buffered saline. Aqueous injectionsuspensions may contain substances which increase the viscosity of thesuspension, such as sodium carboxymethyl cellulose, sorbitol, ordextran. Additionally, suspensions of the active compounds may beprepared as appropriate oily injection suspensions. Suitable lipophilicsolvents or vehicles include fatty oils such as sesame oil, or syntheticfatty acid esters, such as ethyl oleate or triglycerides, or liposomes.Non-lipid polycationic amino polymers may also be used for delivery.Optionally, the suspension may also contain suitable stabilizers oragents which increase the solubility of the compounds to allow for thepreparation of highly concentrated solutions.

[0185] For topical or nasal administration, penetrants appropriate tothe particular barrier to be permeated are used in the formulation. Suchpenetrants are generally known in the art.

[0186] The pharmaceutical compositions of the present invention may bemanufactured in a manner that is known in the art, e.g., by means ofconventional mixing, dissolving, granulating, dragee-making, levigating,emulsifying, encapsulating, entrapping, or lyophilizing processes.

[0187] The pharmaceutical composition may be provided as a salt and canbe formed with many acids, including but not limited to, hydrochloric,sulfuric, acetic, lactic, tartaric, malic, succinic, etc. Salts tend tobe more soluble in aqueous or other protonic solvents than are thecorresponding free base forms. In other cases, the preferred preparationmay be a lyophilized powder which may contain any or all of thefollowing: 1-50 mM histidine, 0.1%-2% sucrose, and 2-7% mannitol, at apH range of 4.5 to 5.5, that is combined with buffer prior to use.

[0188] After pharmaceutical compositions have been prepared, they can beplaced in an appropriate container and labeled for treatment of anindicated condition. For administration of RABP, such labeling wouldinclude amount, frequency, and method of administration.

[0189] Pharmaceutical compositions suitable for use in the inventioninclude compositions wherein the active ingredients are contained in aneffective amount to achieve the intended purpose. The determination ofan effective dose is well within the capability of those skilled in theart.

[0190] For any compound, the therapeutically effective dose can beestimated initially either in cell culture assays, e.g., of neoplasticcells, or in animal models, usually mice, rabbits, dogs, or pigs. Theanimal model may also be used to determine the appropriate concentrationrange and route of administration. Such information can then be used todetermine useful doses and routes for administration in humans.

[0191] A therapeutically effective dose refers to that amount of activeingredient, for example RABP or fragments thereof, antibodies of RABP,agonists, antagonists or inhibitors of RABP, which ameliorates thesymptoms or condition. Therapeutic efficacy and toxicity may bedetermined by standard pharmaceutical procedures in cell cultures orexperimental animals, e.g., ED50 (the dose therapeutically effective in50% of the population) and LD50 (the dose lethal to 50% of thepopulation). The dose ratio between therapeutic and toxic effects is thetherapeutic index, and it can be expressed as the ratio, ED50/LD50.Pharmaceutical compositions which exhibit large therapeutic indices arepreferred. The data obtained from cell culture assays and animal studiesis used in formulating a range of dosage for human use. The dosagecontained in such compositions is preferably within a range ofcirculating concentrations that include the ED50 with little or notoxicity. The dosage varies within this range depending upon the dosageform employed, sensitivity of the patient, and the route ofadministration.

[0192] The exact dosage will be determined by the practitioner, in lightof factors related to the subject that requires treatment. Dosage andadministration are adjusted to provide sufficient levels of the activemoiety or to maintain the desired effect. Factors which may be takeninto account include the severity of the disease state, general healthof the subject, age, weight, and gender of the subject, diet, time andfrequency of administration, drug combination(s), reactionsensitivities, and tolerance/response to therapy. Long-actingpharmaceutical compositions may be administered every 3 to 4 days, everyweek, or once every two weeks depending on half-life and clearance rateof the particular formulation.

[0193] Normal dosage amounts may vary from 0.1 to 100,000 micrograms, upto a total dose of about 1 g, depending upon the route ofadministration. Guidance as to particular dosages and methods ofdelivery is provided in the literature and generally available topractitioners in the art. Those skilled in the art will employ differentformulations for nucleotides than for proteins or their inhibitors.Similarly, delivery of polynucleotides or polypeptides will be specificto particular cells, conditions, locations, etc.

[0194] Diagnostics

[0195] In another embodiment, antibodies which specifically bind RABPmay be used for the diagnosis of conditions or diseases characterized byexpression of RABP, or in assays to monitor patients being treated withRABP, agonists, antagonists or inhibitors. The antibodies useful fordiagnostic purposes may be prepared in the same manner as thosedescribed above for therapeutics. Diagnostic assays for RABP includemethods which utilize the antibody and a label to detect RABP in humanbody fluids or extracts of cells or tissues. The antibodies may be usedwith or without modification, and may be labeled by joining them, eithercovalently or non-covalently, with a reporter molecule. A wide varietyof reporter molecules which are known in the art may be used, several ofwhich are described above.

[0196] A variety of protocols including ELISA, RIA, and FACS formeasuring RABP are known in the art and provide a basis for diagnosingaltered or abnormal levels of RABP expression. Normal or standard valuesfor RABP expression are established by combining body fluids or cellextracts taken from normal mammalian subjects, preferably human, withantibody to RABP under conditions suitable for complex formation. Theamount of standard complex formation may be quantified by variousmethods, but preferably by photometric means. Quantities of RABPexpressed in subject samples, control and disease, from biopsied tissuesare compared with the standard values. Deviation between standard andsubject values establishes the parameters for diagnosing disease.

[0197] In another embodiment of the invention, the polynucleotidesencoding RABP may be used for diagnostic purposes. The polynucleotideswhich may be used include oligonucleotide sequences, complementary RNAand DNA molecules, and PNAs. The polynucleotides may be used to detectand quantitate gene expression in biopsied tissues in which expressionof RABP may be correlated with disease. The diagnostic assay may be usedto distinguish between absence, presence, and excess expression of RABP,and to monitor regulation of RABP levels during therapeuticintervention.

[0198] In one aspect, hybridization with PCR probes which are capable ofdetecting polynucleotide sequences, including genomic sequences,encoding RABP or closely related molecules, may be used to identifynucleic acid sequences which encode RABP. The specificity of the probe,whether it is made from a highly specific region, e.g., 10 uniquenucleotides in the 5′ regulatory region, or a less specific region,e.g., especially in the 3′ coding region, and the stringency of thehybridization or amplification (maximal, high, intermediate, or low)will determine whether the probe identifies only naturally occurringsequences encoding RABP, alleles, or related sequences.

[0199] Probes may also be used for the detection of related sequences,and should preferably contain at least 50% of the nucleotides from anyof the RABP encoding sequences. The hybridization probes of the subjectinvention may be DNA or RNA and derived from the nucleotide sequence ofSEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, or from genomic sequenceincluding promoter, enhancer elements, and introns of the naturallyoccurring RABP.

[0200] Means for producing specific hybridization probes for DNAsencoding RABP include the cloning of nucleic acid sequences encodingRABP or RABP derivatives into vectors for the production of mRNA probes.Such vectors are known in the art, commercially available, and may beused to synthesize RNA probes in vitro by means of the addition of theappropriate RNA polymerases and the appropriate labeled nucleotides.Hybridization probes may be labeled by a variety of reporter groups, forexample, radionuclides such as 32P or 35S, or enzymatic labels, such asalkaline phosphatase coupled to the probe via avidin/biotin couplingsystems, and the like.

[0201] Polynucleotide sequences encoding RABP may be used for thediagnosis of conditions or disorders which are associated withexpression of RABP. Examples of such conditions or disorders include,but are not limited to, disorders associated with cell proliferationsuch as adenocarcinoma, leukemia, lymphoma, melanoma, myeloma, sarcoma,and teratocarcinoma, and particularly, cancers of the adrenal gland,bladder, bone, bone marrow, brain, breast, cervix, gall bladder,ganglia, gastrointestinal tract, heart, kidney, liver, lung, muscle,ovary, pancreas, parathyroid, penis, prostate, salivary glands, skin,spleen, testis, thymus, thyroid, and uterus; disorders with associatedinflammation such as Addison's disease, adult respiratory distresssyndrome, allergies, anemia, asthma, atherosclerosis, bronchitis,cholecystitus, Crohn's disease, ulcerative colitis, atopic dermatitis,dermatomyositis, diabetes mellitus, emphysema, atrophic gastritis,glomerulonephritis, gout, Graves' disease, hypereosinophilia, irritablebowel syndrome, lupus erythematosus, multiple sclerosis, myastheniagravis, myocardial or pericardial inflammation,osteoarthritis,osteoporosis, pancreatitis, polymyositis, rheumatoid arthritis,scleroderma, Sjögren's syndrome, and autoimmune thyroiditis;complications of cancer, hemodialysis, extracorporeal circulation;viral, bacterial, fungal, parasitic, protozoal, and helminthicinfections and trauma; disorders with associated apoptosis such as AIDSand other infectious or genetic immunodeficiencies, neurodegenerativediseases such as Alzheimer's disease, Parkinson's disease, amyotrophiclateral sclerosis, retinitis pigmentosa, and cerebellar degeneration,myelodysplastic syndromes such as aplastic anemia, ischemic injuriessuch as myocardial infarction, stroke, and reperfusion injury,toxin-induced diseases such as alcohol-induced liver damage, cirrhosis,and lathyrism, wasting diseases such as cachexia, viral infections suchas those caused by hepatitis B and C, and osteoporosis. Thepolynucleotide sequences encoding RABP may be used in Southern ornorthern analysis, dot blot, or other membrane-based technologies; inPCR technologies; or in dipstick, pin, ELISA assays or microarraysutilizing fluids or tissues from patient biopsies to detect altered RABPexpression. Such qualitative or quantitative methods are well known inthe art.

[0202] In a particular aspect, the nucleotide sequences encoding RABPmay be useful in assays that detect activation or induction of variouscancers, particularly those mentioned above. The nucleotide sequencesencoding RABP may be labeled by standard methods, and added to a fluidor tissue sample from a patient under conditions suitable for theformation of hybridization complexes. After a suitable incubationperiod, the sample is washed and the signal is quantitated and comparedwith a standard value. If the amount of signal in the biopsied orextracted sample is significantly altered from that of a comparablecontrol sample, the nucleotide sequences have hybridized with nucleotidesequences in the sample, and the presence of altered levels ofnucleotide sequences encoding RABP in the sample indicates the presenceof the associated disease. Such assays may also be used to evaluate theefficacy of a particular therapeutic treatment regimen in animalstudies, in clinical trials, or in monitoring the treatment of anindividual patient.

[0203] In order to provide a basis for the diagnosis of diseaseassociated with expression of RABP, a normal or standard profile forexpression is established. This may be accomplished by combining bodyfluids or cell extracts taken from normal subjects, either animal orhuman, with a sequence, or a fragment thereof, which encodes RABP, underconditions suitable for hybridization or amplification. Standardhybridization may be quantified by comparing the values obtained fromnormal subjects with those from an experiment where a known amount of asubstantially purified polynucleotide is used. Standard values obtainedfrom normal samples may be compared with values obtained from samplesfrom patients who are symptomatic for disease. Deviation betweenstandard and subject values is used to establish the presence ofdisease.

[0204] Once disease is established and a treatment protocol isinitiated, hybridization assays may be repeated on a regular basis toevaluate whether the level of expression in the patient begins toapproximate that which is observed in the normal patient. The resultsobtained from successive assays may be used to show the efficacy oftreatment over a period ranging from several days to months.

[0205] With respect to cancer, the presence of a relatively high amountof transcript in biopsied tissue from an individual may indicate apredisposition for the development of the disease, or may provide ameans for detecting the disease prior to the appearance of actualclinical symptoms. A more definitive diagnosis of this type may allowhealth professionals to employ preventative measures or aggressivetreatment earlier thereby preventing the development or furtherprogression of the cancer.

[0206] Additional diagnostic uses for oligonucleotides designed from thesequences encoding RABP may involve the use of PCR. Such oligomers maybe chemically synthesized, generated enzymatically, or produced invitro. Oligomers will preferably consist of two nucleotide sequences,one with sense orientation (5′→3′) and another with antisense (3′→5′),employed under optimized conditions for identification of a specificgene or condition. The same two oligomers, nested sets of oligomers, oreven a degenerate pool of oligomers may be employed under less stringentconditions for detection and/or quantitation of closely related DNA orRNA sequences.

[0207] Methods which may also be used to quantitate the expression ofRABP include radiolabeling or biotinylating nucleotides, coamplificationof a control nucleic acid, and standard curves onto which theexperimental results are interpolated (Melby, P. C. et al. (1993) J.Immunol. Methods, 159:235-244; Duplaa, C. et al. (1993) Anal. Biochem.229-236). The speed of quantitation of multiple samples may beaccelerated by running the assay in an ELISA format where the oligomerof interest is presented in various dilutions and a spectrophotometricor colorimetric response gives rapid quantitation.

[0208] In further embodiments, an oligonucleotide derived from any ofthe polynucleotide sequences described herein may be used as a target ina microarray. The microarray can be used to monitor the expression levelof large numbers of genes simultaneously (to produce a transcriptimage), and to identify genetic variants, mutations and polymorphisms.This information will be useful in determining gene function,understanding the genetic basis of disease, diagnosing disease, and indeveloping and monitoring the activity of therapeutic agents (Heller, R.et al. (1997) Proc. Natl. Acad. Sci. 94:2150-55).

[0209] In one embodiment, the microarray is prepared and used accordingto the methods described in PCT application WO95/11995 (Chee et al.),Lockhart, D. J. et al. (1996; Nat. Biotech. 14: 1675-1680) and Schena,M. et al. (1996; Proc. Natl. Acad. Sci. 93: 10614-10619), all of whichare incorporated herein in their entirety by reference.

[0210] The microarray is preferably composed of a large number ofunique, single-stranded nucleic acid sequences, usually either syntheticantisense oligonucleotides or fragments of cDNAs, fixed to a solidsupport. The oligonucleotides are preferably about 6-60 nucleotides inlength, more preferably 15-30 nucleotides in length, and most preferablyabout 20-25 nucleotides in length. For a certain type of microarray, itmay be preferable to use oligonucleotides which are only 7-10nucleotides in length. The microarray may contain oligonucleotides whichcover the known 5′, or 3′, sequence, sequential oligonucleotides whichcover the full length sequence; or unique oligonucleotides selected fromparticular areas along the length of the sequence. Polynucleotides usedin the microarray may be oligonucleotides that are specific to a gene orgenes of interest in which at least a fragment of the sequence is knownor that are specific to one or more unidentified cDNAs which are commonto a particular cell type, developmental or disease state.

[0211] In order to produce oligonucleotides to a known sequence for amicroarray, the gene of interest is examined using a computer algorithmwhich starts at the 5′ or more preferably at the 3′ end of thenucleotide sequence. The algorithm identifies oligomers of definedlength that are unique to the gene, have a GC content within a rangesuitable for hybridization, and lack predicted secondary structure thatmay interfere with hybridization. In certain situations it may beappropriate to use pairs of oligonucleotides on a microarray. The“pairs” will be identical, except for one nucleotide which preferably islocated in the center of the sequence. The second oligonucleotide in thepair (mismatched by one) serves as a control. The number ofoligonucleotide pairs may range from two to one million. The oligomersare synthesized at designated areas on a substrate using alight-directed chemical process. The substrate may be paper, nylon orother type of membrane, filter, chip, glass slide or any other suitablesolid support.

[0212] In another aspect, an oligonucleotide may be synthesized on thesurface of the substrate by using a chemical coupling procedure and anink jet application apparatus, as described in PCT applicationWO95/251116 (Baldeschweiler et al.) which is incorporated herein in itsentirety by reference. In another aspect, a “gridded” array analogous toa dot (or slot) blot may be used to arrange and link cDNA fragments oroligonucleotides to the surface of a substrate using a vacuum system,thermal, UV, mechanical or chemical bonding procedures. An array, suchas those described above, may be produced by hand or by using availabledevices (slot blot or dot blot apparatus), materials (any suitable solidsupport), and machines (including robotic instruments), and may contain8, 24, 96, 384, 1536 or 6144 oligonucleotides, or any other numberbetween two and one million which lends itself to the efficient use ofcommercially available instrumentation.

[0213] In order to conduct sample analysis using a microarray, the RNAor DNA from a biological sample is made into hybridization probes. ThemRNA is isolated, and cDNA is produced and used as a template to makeantisense RNA (aRNA). The aRNA is amplified in the presence offluorescent nucleotides, and labeled probes are incubated with themicroarray so that the probe sequences hybridize to complementaryoligonucleotides of the microarray. Incubation conditions are adjustedso that hybridization occurs with precise complementary matches or withvarious degrees of less complementarity. After removal of nonhybridizedprobes, a scanner is used to determine the levels and patterns offluorescence. The scanned images are examined to determine degree ofcomplementarity and the relative abundance of each oligonucleotidesequence on the microarray. The biological samples may be obtained fromany bodily fluids (such as blood, urine, saliva, phlegm, gastric juices,etc.), cultured cells, biopsies, or other tissue preparations. Adetection system may be used to measure the absence, presence, andamount of hybridization for all of the distinct sequencessimultaneously. This data may be used for large scale correlationstudies on the sequences, mutations, variants, or polymorphisms amongsamples.

[0214] In another embodiment of the invention, the nucleic acidsequences which encode RABP may also be used to generate hybridizationprobes which are useful for mapping the naturally occurring genomicsequence. The sequences may be mapped to a particular chromosome, to aspecific region of a chromosome or to artificial chromosomeconstructions, such as human artificial chromosomes (HACs), yeastartificial chromosomes (YACs), bacterial artificial chromosomes (BACs),bacterial P1 constructions or single chromosome cDNA libraries asreviewed in Price, C. M. (1993) Blood Rev. 7:127-134, and Trask, B. J.(1991) Trends Genet. 7:149-154.

[0215] Fluorescent in situ hybridization (FISH as described in Verma etal. (1988) Human Chromosomes: A Manual of Basic Techniques, PergamonPress, New York, N.Y.) may be correlated with other physical chromosomemapping techniques and genetic map data. Examples of genetic map datacan be found in various scientific journals or at Online MendelianInheritance in Man (OMIM). Correlation between the location of the geneencoding RABP on a physical chromosomal map and a specific disease, orpredisposition to a specific disease, may help delimit the region of DNAassociated with that genetic disease. The nucleotide sequences of thesubject invention may be used to detect differences in gene sequencesbetween normal, carrier, or affected individuals.

[0216] In situ hybridization of chromosomal preparations and physicalmapping techniques such as linkage analysis using establishedchromosomal markers may be used for extending genetic maps. Often theplacement of a gene on the chromosome of another mammalian species, suchas mouse, may reveal associated markers even if the number or arm of aparticular human chromosome is not known. New sequences can be assignedto chromosomal arms, or parts thereof, by physical mapping. Thisprovides valuable information to investigators searching for diseasegenes using positional cloning or other gene discovery techniques. Oncethe disease or syndrome has been crudely localized by genetic linkage toa particular genomic region, for example, AT to 11q22-23 (Gatti, R. A.et al. (1988) Nature 336:577-580), any sequences mapping to that areamay represent associated or regulatory genes for further investigation.The nucleotide sequence of the subject invention may also be used todetect differences in the chromosomal location due to translocation,inversion, etc. among normal, carrier, or affected individuals.

[0217] In another embodiment of the invention, RABP, its catalytic orimmunogenic fragments or oligopeptides thereof, can be used forscreening libraries of compounds in any of a variety of drug screeningtechniques. The fragment employed in such screening may be free insolution, affixed to a solid support, borne on a cell surface, orlocated intracellularly. The formation of binding complexes, betweenRABP and the agent being tested, may be measured.

[0218] Another technique for drug screening which may be used providesfor high throughput screening of compounds having suitable bindingaffinity to the protein of interest as described in published PCTapplication WO84/03564. In this method, as applied to RABP, largenumbers of different small test compounds are synthesized on a solidsubstrate, such as plastic pins or some other surface. The testcompounds are reacted with RABP, or fragments thereof, and washed. BoundRABP is then detected by methods well known in the art. Purified RABPcan also be coated directly onto plates for use in the aforementioneddrug screening techniques. Alternatively, non-neutralizing antibodiescan be used to capture the peptide and immobilize it on a solid support.

[0219] In another embodiment, one may use competitive drug screeningassays in which neutralizing antibodies capable of binding RABPspecifically compete with a test compound for binding RABP. In thismanner, the antibodies can be used to detect the presence of any peptidewhich shares one or more antigenic determinants with RABP.

[0220] In additional embodiments, the nucleotide sequences which encodeRABP may be used in any molecular biology techniques that have yet to bedeveloped, provided the new techniques rely on properties of nucleotidesequences that are currently known, including, but not limited to, suchproperties as the triplet genetic code and specific base pairinteractions.

[0221] The examples below are provided to illustrate the subjectinvention and are not included for the purpose of limiting theinvention.

EXAMPLES

[0222] I cDNA Library Construction

[0223] The NGANNOT01 cDNA library was constructed using 1 microgram ofpolyA RNA isolated from tumorous neuroganglion tissue removed from a9-year-old Caucasian male during a soft tissue excision of the chestwall. Pathology indicated a ganglioneuroma forming an encapsulatedlobulated mass. Examination of the medial aspect of the pleurasurrounding the tumor showed fibrotic tissue with chronic inflammationthat extended into the overlying adipose tissue. The patient presentedwith a cough and was not taking any medications.

[0224] The LIVRTUT04 cDNA library was constructed using 7.5 nanograms ofpolyA RNA isolated from liver tumor tissue removed from a 50-year-oldCaucasian male during a partial hepatectomy. Pathology indicated a grade3-4 hepatoma; surgical margins free of tumor; and no lymphovascularinvasion. The adjacent liver showed mild portal fibrosis with lymphoidaggregates and mild steatosis. Patient history included benignhypertension and hepatitis.

[0225] The UTRSNOT16 cDNA library was constructed using 2 micrograms ofpolyA RNA isolated from nontumorous uterine endometrial tissue removedfrom a 48-year-old Caucasian female during a vaginal hysterectomy,rectocele repair, and bilateral salpingo-oopherectomy. Pathologyindicated chronic cervicitis, and the endometrium was weaklyproliferative. The uterus, tubes, ovaries, and specimen from theperitoneum indicated endometriosis focally involving the surface of theright ovary and the peritoneum. Pathology for the associated tumortissue indicated a single submucosal leiomyoma, which exhibitedextensive hyalin change with hyalin-type necrosis. The left ovarycontained a corpus luteum cyst, and the right and left fallopian tubeswere unremarkable. The patient presented with metrorrhagia, extrinsicasthma, depressive disorder, and anxiety.

[0226] The frozen tissues were homogenized and lysed using a BrinkmannHomogenizer Polytron PT-3000 (Brinkmann Instruments, Westbury, N.J.) inguanidinium isothiocyanate solution. The lysates were centrifuged over a5.7 M CsCl cushion using a Beckman SW28 rotor in a Beckman L8-70MUltracentrifuge (Beckman Instruments) for 18 hours at 25,000 rpm atambient temperature. The RNA was extracted with acid phenol pH 4.7,precipitated using 0.3 M sodium acetate and 2.5 volumes of ethanol,resuspended in RNAse-free water, and DNase treated at 37° C. RNAextraction and precipitation were repeated as before. The mRNA was thenisolated using the Qiagen OLIGOTEX kit (QIAGEN, Chatsworth, Calif.) andused to construct the cDNA libraries.

[0227] The mRNAs were handled according to the recommended protocols inthe SuperScript Plasmid System for cDNA Synthesis and Plasmid Cloning(Cat. #18248-013, Gibco/BRL). cDNAs were fractionated on a SEPHAROSECL4B column (Cat. #275105-01, Pharmacia), and those cDNAs exceeding 400bp were ligated into PSPORT1 (NGANNOT01) or pINCY (LIVRTUT04 andUTRSNOT16). The plasmid PSPORT1 or pINCY were subsequently transformedinto DH5α competent cells (Cat. #18258-012, Gibco/BRL).

[0228] II Isolation and Sequencing of cDNA Clones

[0229] Plasmid DNA was released from the cells and purified using theREAL Prep 96 Plasmid Kit (Catalog #26173, QIAGEN). This kit enabled thesimultaneous purification of 96 samples in a 96-well block usingmulti-channel reagent dispensers. The recommended protocol was employedexcept for the following changes: 1) the bacteria were cultured in 1 mlof sterile Terrific Broth (Catalog #22711, GIBCO/BRL) with carbenicillinat 25 mg/L and glycerol at 0.4%; 2) after inoculation, the cultures wereincubated for 19 hours and at the end of incubation, the cells werelysed with 0.3 ml of lysis buffer; and 3) following isopropanolprecipitation, the plasmid DNA pellet was resuspended in 0.1 ml ofdistilled water. After the last step in the protocol, samples weretransferred to a 96-well block for storage at 4° C.

[0230] The cDNAs were sequenced by the method of Sanger et al. (1975, J.Mol. Biol. 94:441f), using a Hamilton Micro Lab 2200 (Hamilton, Reno,Nev.) in combination with Peltier Thermal Cyclers (PTC200 from MJResearch, Watertown, Mass.) and Applied Biosystems 377 DNA SequencingSystems; and the reading frame was determined.

[0231] III Homology Searching of cDNA Clones and Their Deduced Proteins

[0232] The nucleotide sequences of the Sequence Listing or amino acidsequences deduced from them were used as query sequences againstdatabases such as GenBank, SwissProt, BLOCKS, and Pima II. Thesedatabases which contain previously identified and annotated sequenceswere searched for regions of homology (similarity) using BLAST, whichstands for Basic Local Alignment Search Tool (Altschul S F (1993) J.Mol. Evol. 36:290-300; Altschul, S F et al. (1990) J. Mol. Biol.215:403-10).

[0233] BLAST produces alignments of both nucleotide and amino acidsequences to determine sequence similarity. Because of the local natureof the alignments, BLAST is especially useful in determining exactmatches or in identifying homologs which may be of prokaryotic(bacterial) or eukaryotic (animal, fungal or plant) origin. Otheralgorithms such as the one described in Smith R F and T F Smith (1992Protein Engineering 5:35-51), incorporated herein by reference, can beused when dealing with primary sequence patterns and secondary structuregap penalties. As disclosed in this application, the sequences havelengths of at least 49 nucleotides, and no more than 12% uncalled bases(where N is recorded rather than A, C, G, or T).

[0234] The BLAST approach, as detailed in Karlin and Altschul (1993;Proc Nat Acad Sci 90:5873-7) and incorporated herein by reference,searches matches between a query sequence and a database sequence, toevaluate the statistical significance of any matches found, and toreport only those matches which satisfy the user-selected threshold ofsignificance. In this application, threshold was set at 10-25 fornucleotides and 10-14 for peptides.

[0235] IV Northern Analysis

[0236] Northern analysis is a laboratory technique used to detect thepresence of a transcript of a gene and involves the hybridization of alabeled nucleotide sequence to a membrane on which RNAs from aparticular cell type or tissue have been bound (Sambrook et al., supra).

[0237] Analogous computer techniques using BLAST (Altschul, S. F. (1993)J.Mol.Evol. 36:290-300; Altschul, S. F. et al. (1990) J.Mol.Evol.215:403-410) are used to search for identical or related molecules innucleotide databases such as GenBank or the LIFESEQ database (IncytePharmaceuticals). This analysis is much faster than multiple,membrane-based hybridizations. In addition, the sensitivity of thecomputer search can be modified to determine whether any particularmatch is categorized as exact or homologous.

[0238] The basis of the search is the product score which is defined as:

[0239] % sequence identity×% maximum BLAST score/100

[0240] The product score takes into account both the degree ofsimilarity between two sequences and the length of the sequence match.For example, with a product score of 40, the match will be exact withina 1-2% error; and at 70, the match will be exact. Homologous moleculesare usually identified by selecting those which show product scoresbetween 15 and 40, although lower scores may identify related molecules.

[0241] The results of northern analysis are reported as a list oflibraries in which the transcript encoding RABP occurs. Abundance andpercent abundance are also reported. Abundance directly reflects thenumber of times a particular transcript is represented in a cDNAlibrary, and percent abundance is abundance divided by the total numberof sequences examined in the cDNA library.

[0242] V Extension of RABP Encoding Polynucleotides

[0243] The nucleic acid sequence of the Incyte Clone 2312652, 2514506,or 3400003 was used to design oligonucleotide primers for extending apartial nucleotide sequence to full length. One primer was synthesizedto initiate extension in the antisense direction, and the other wassynthesized to extend sequence in the sense direction. Primers were usedto facilitate the extension of the known sequence “outward” generatingamplicons containing new, unknown nucleotide sequence for the region ofinterest. The initial primers were designed from the cDNA using OLIGO4.06 (National Biosciences), or another appropriate program, to be about22 to about 30 nucleotides in length, to have a GC content of 50% ormore, and to anneal to the target sequence at temperatures of about 68°to about 72° C. Any stretch of nucleotides which would result in hairpinstructures and primer-primer dimerizations was avoided.

[0244] Selected human cDNA libraries (Gibco/BRL) were used to extend thesequence. If more than one extension is necessary or desired, additionalsets of primers are designed to further extend the known region.

[0245] High fidelity amplification was obtained by following theinstructions for the XL-PCR kit (Perkin Elmer) and thoroughly mixing theenzyme and reaction mix. Beginning with 40 pmol of each primer and therecommended concentrations of all other components of the kit, PCR wasperformed using the Peltier Thermal Cycler (PTC200; M.J. Research,Watertown, Mass.) and the following parameters: Step 1 94° C. for 1 min(initial denaturation) Step 2 65° C. for 1 min Step 3 68° C. for 6 minStep 4 94° C. for 15 sec Step 5 65° C. for 1 min Step 6 68° C. for 7 minStep 7 Repeat step 4-6 for 15 additional cycles Step 8 94° C. for 15 secStep 9 65° C. for 1 min Step 10 68° C. for 7:15 min Step 11 Repeat step8-10 for 12 cycles Step 12 72° C. for 8 min Step 13 4° C. (and holding)

[0246] A 5-10 μl aliquot of the reaction mixture was analyzed byelectrophoresis on a low concentration (about 0.6-0.8%) agarose mini-gelto determine which reactions were successful in extending the sequence.Bands thought to contain the largest products were excised from the gel,purified using QIAQIUK (QIAGEN Inc., Chatsworth, Calif.), and trimmed ofoverhangs using Klenow enzyme to facilitate religation and cloning.

[0247] After ethanol precipitation, the products were redissolved in 13μl of ligation buffer, 1 μl T4-DNA ligase (15 units) and 1 μl T4polynucleotide kinase were added, and the mixture was incubated at roomtemperature for 2-3 hours or overnight at 16° C. Competent E. coli cells(in 40 μl of appropriate media) were transformed with 3 μl of ligationmixture and cultured in 80 μl of SOC medium (Sambrook et al., supra).After incubation for one hour at 37° C., the E. coli mixture was platedon Luria Bertani (LB)-agar (Sambrook et al., supra) containing 2× Carb.The following day, several colonies were randomly picked from each plateand cultured in 150 μl of liquid LB/2× Carb medium placed in anindividual well of an appropriate, commercially-available, sterile96-well microtiter plate. The following day, 5 μl of each overnightculture was transferred into a non-sterile 96-well plate and afterdilution 1:10 with water, 5 μl of each sample was transferred into a PCRarray.

[0248] For PCR amplification, 18 μl of concentrated PCR reaction mix(3.3×) containing 4 units of rTth DNA polymerase, a vector primer, andone or both of the gene specific primers used for the extension reactionwere added to each well. Amplification was performed using the followingconditions: Step 1 94° C. for 60 sec Step 2 94° C. for 20 sec Step 3 55°C. for 30 sec Step 4 72° C. for 90 sec Step 5 Repeat steps 2-4 for anadditional 29 cycles Step 6 72° C. for 180 sec Step 7 4° C. (andholding)

[0249] Aliquots of the PCR reactions were run on agarose gels togetherwith molecular weight markers. The sizes of the PCR products werecompared to the original partial cDNAs, and appropriate clones wereselected, ligated into plasmid, and sequenced.

[0250] In like manner, the nucleotide sequence of SEQ ID NO:2, SEQ IDNO:4, or SEQ ID NO:6, is used to obtain 5′ regulatory sequences usingthe procedure above, oligonucleotides designed for 5′ extension, and anappropriate genomic library.

[0251] VI Labeling and Use of Individual Hybridization Probes

[0252] Hybridization probes derived from SEQ ID NO:2, SEQ ID NO:4, orSEQ ID NO:6 are employed to screen cDNAs, genomic DNAs, or mRNAs.Although the labeling of oligonucleotides, consisting of about 20base-pairs, is specifically described, essentially the same procedure isused with larger nucleotide fragments. Oligonucleotides are designedusing state-of-the-art software such as OLIGO 4.06 (NationalBiosciences), labeled by combining 50 pmol of each oligomer and 250 μCiof [γ-⁼P] adenosine triphosphate (Amersham) and T4 polynucleotide kinase(DuPont NEN, Boston, Mass.). The labeled oligonucleotides aresubstantially purified with SEPHADEX G-25 superfine resin column(Pharmacia & Upjohn). A aliquot containing 10⁷ counts per minute of thelabeled probe is used in a typical membrane-based hybridization analysisof human genomic DNA digested with one of the following endonucleases(Ase I, Bg1 II, Eco RI, Pst I, Xba 1, or Pvu II; DuPont NEN).

[0253] The DNA from each digest is fractionated on a 0.7 percent agarosegel and transferred to nylon membranes (Nytran Plus, Schleicher &Schuell, Durham, N.H.). Hybridization is carried out for 16 hours at 40°C. To remove nonspecific signals, blots are sequentially washed at roomtemperature under increasingly stringent conditions up to 0.1× salinesodium citrate and 0.5% sodium dodecyl sulfate. After XOMAT AR film(Kodak, Rochester, N.Y.) is exposed to the blots in a Phosphoimagercassette (Molecular Dynamics, Sunnyvale, Calif.) for several hours,hybridization patterns are compared visually.

[0254] VII Microarrays

[0255] To produce oligonucleotides for a microarray, the nucleotidesequence described herein is examined using a computer algorithm whichstarts at the 3′ end of the nucleotide sequence. The algorithmidentifies oligomers of defined length that are unique to the gene, havea GC content within a range suitable for hybridization, and lackpredicted secondary structure that would interfere with hybridization.The algorithm identifies 20 sequence-specific oligonucleotides of 20nucleotides in length (20-mers). A matched set of oligonucleotides iscreated in which one nucleotide in the center of each sequence isaltered. This process is repeated for each gene in the microarray, anddouble sets of twenty 20 mers are synthesized and arranged on thesurface of the silicon chip using a light-directed chemical process(Chee, M. et al., PCT/WO95/11995, incorporated herein by reference).

[0256] In the alternative, a chemical coupling procedure and an ink jetdevice are used to synthesize oligomers on the surface of a substrate(Baldeschweiler, J. D. et al., PCT/WO95/25116, incorporated herein byreference). In another alternative, a “gridded” array analogous to a dot(or slot) blot is used to arrange and link cDNA fragments oroligonucleotides to the surface of a substrate using a vacuum system,thermal, UV, mechanical or chemical bonding procedures. An array may beproduced by hand or using available materials and machines and containgrids of 8 dots, 24 dots, 96 dots, 384 dots, 1536 dots or 6144 dots.After hybridization, the microarray is washed to remove nonhybridizedprobes, and a scanner is used to determine the levels and patterns offluorescence. The scanned images are examined to determine degree ofcomplementarity and the relative abundance of each oligonucleotidesequence on the micro-array.

[0257] VIII Complementary Polynucleotides

[0258] Sequence complementary to the RABP-encoding sequence, or any partthereof, is used to decrease or inhibit expression of naturallyoccurring RABP. Although use of oligonucleotides comprising from about15 to about 30 base-pairs is described, essentially the same procedureis used with smaller or larger sequence fragments. Appropriateoligonucleotides are designed using Oligo 4.06 software and the codingsequence of RABP, SEQ ID NO:1, SEQ ID NO:3, or SEQ ID NO:5. To inhibittranscription, a complementary oligonucleotide is designed from the mostunique 5′ sequence and used to prevent promoter binding to the codingsequence. To inhibit translation, a complementary oligonucleotide isdesigned to prevent ribosomal binding to the RABP-encoding transcript.

[0259] IX Expression of RABP

[0260] Expression of RABP is accomplished by subcloning the cDNAs intoappropriate vectors and transforming the vectors into host cells. Inthis case, the cloning vector is also used to express RABP in E. coli.Upstream of the cloning site, this vector contains a promoter forβ-galactosidase, followed by sequence containing the amino-terminal Met,and the subsequent seven residues of β-galactosidase. Immediatelyfollowing these eight residues is a bacteriophage promoter useful fortranscription and a linker containing a number of unique restrictionsites.

[0261] Induction of an isolated, transformed bacterial strain with IPTGusing standard methods produces a fusion protein which consists of thefirst eight residues of β-galactosidase, about 5 to 15 residues oflinker, and the full length protein. The signal residues direct thesecretion of RABP into the bacterial growth media which can be useddirectly in the following assay for activity.

[0262] X Demonstration of RABP Activity

[0263] RABP can be expressed in a mammalian cell line such as 293T bytransfecting with an eukaryotic expression vector encoding RABP.Eukaryotic expression vectors are commercially available, and thetechniques to introduce them into cells are well known to those skilledin the art. A small amount of a second plasmid, which expresses any oneof a number of reporter genes such as β-galactosidase, is co-transformedinto the cells in order to allow rapid identification of those cellswhich have taken up and expressed the foreign DNA. The cells arecultured in a defined synthetic medium with concentrations of GTP for atleast 48 hours after transformation to allow expression and accumulationof RABP and β-galactosidase.

[0264] Transformed cells expressing β-galactosidase are stained bluewhen a suitable colorimetric substrate is added to the culture mediaunder conditions that are well known in the art. Increasingconcentrations of GTP induces increasing numbers of reporter genepositive cells (Ren, M. et al. (1996) Proc. Natl. Acad. Sci. 93:5151-5155). GTP-treated cells which were not transformed with the RABPexpression vector are used as controls as are RABP transfected cellscultured without supplemental GTP.

[0265] XI Production of RABP Specific Antibodies

[0266] RABP that is substantially purified using PAGE electrophoresis(Sambrook, supra), or other purification techniques, is used to immunizerabbits and to produce antibodies using standard protocols. The aminoacid sequence deduced from SEQ ID NO:2, SEQ ID NO:4, or SEQ ID NO:6 isanalyzed using DNASTAR software (DNASTAR Inc) to determine regions ofhigh immunogenicity and a corresponding oligopeptide is synthesized andused to raise antibodies by means known to those of skill in the art.Selection of appropriate epitopes, such as those near the C-terminus orin hydrophilic regions, is described by Ausubel et al. (supra), andothers.

[0267] Typically, the oligopeptides are 15 residues in length,synthesized using an Applied Biosystems Peptide Synthesizer Model 43 1Ausing fmoc-chemistry, and coupled to keyhole limpet hemocyanin (KLH,Sigma, St. Louis, Mo.) by reaction withN-maleimidobenzoyl-N-hydroxysuccinimide ester (MBS; Ausubel et al.,supra). Rabbits are immunized with the oligopeptide-KLH complex incomplete Freund's adjuvant. The resulting antisera are tested forantipeptide activity, for example, by binding the peptide to plastic,blocking with 1% BSA, reacting with rabbit antisera, washing, andreacting with radio iodinated, goat anti-rabbit IgG.

[0268] XII Purification of Naturally Occurring RABP Using SpecificAntibodies

[0269] Naturally occurring or recombinant RABP is substantially purifiedby immunoaffinity chromatography using antibodies specific for RABP. Animmunoaffinity column is constructed by covalently coupling RABPantibody to an activated chromatographic resin, such as CNBr-activatedSEPHAROSE (Pharmacia & Upjohn). After the coupling, the resin is blockedand washed according to the manufacturer's instructions.

[0270] Media containing RABP is passed over the immunoaffinity column,and the column is washed under conditions that allow the preferentialabsorbance of RABP (e.g., high ionic strength buffers in the presence ofdetergent). The column is eluted under conditions that disruptantibody/RABP binding (eg, a buffer of pH 2-3 or a high concentration ofa chaotrope, such as urea or thiocyanate ion), and RABP is collected.

[0271] XIII Identification of Molecules Which Interact with RABP

[0272] RABP or biologically active fragments thereof are labeled with¹²⁵I Bolton-Hunter reagent (Bolton et al. (1973) Biochem. J. 133: 529).Candidate molecules previously arrayed in the wells of a multi-wellplate are incubated with the labeled RABP, washed and any wells withlabeled RABP complex are assayed. Data obtained using differentconcentrations of RABP are used to calculate values for the number,affinity, and association of RABP with the candidate molecules.

[0273] All publications and patents mentioned in the above specificationare herein incorporated by reference. Various modifications andvariations of the described method and system of the invention will beapparent to those skilled in the art without departing from the scopeand spirit of the invention. Although the invention has been describedin connection with specific preferred embodiments, it should beunderstood that the invention as claimed should not be unduly limited tosuch specific embodiments. Indeed, various modifications of thedescribed modes for carrying out the invention which are obvious tothose skilled in molecular biology or related fields are intended to bewithin the scope of the following claims.

1 9 203 amino acids amino acid single linear NGANNOT01 2312652 1 Met SerGly Gln Arg Val Asp Val Lys Val Val Met Leu Gly Lys Glu 1 5 10 15 TyrVal Gly Lys Thr Ser Leu Val Glu Arg Tyr Val His Xaa Xaa Phe 20 25 30 LeuVal Gly Pro Tyr Gln Asn Thr Ile Gly Ala Ala Phe Val Ala Lys 35 40 45 ValMet Ser Val Gly Asp Arg Thr Val Thr Leu Gly Ile Trp Asp Thr 50 55 60 AlaGly Ser Glu Arg Tyr Glu Ala Met Ser Arg Ile Tyr Tyr Arg Gly 65 70 75 80Ala Lys Ala Ala Ile Val Cys Tyr Asp Leu Thr Asp Ser Ser Ser Phe 85 90 95Glu Arg Ala Lys Phe Trp Val Lys Glu Leu Arg Ser Leu Glu Glu Gly 100 105110 Cys Gln Ile Tyr Leu Cys Gly Thr Lys Ser Asp Leu Leu Glu Glu Asp 115120 125 Arg Arg Arg Arg Arg Val Asp Phe His Asp Val Gln Asp Tyr Ala Asp130 135 140 Asn Ile Lys Ala Gln Leu Phe Glu Thr Ser Ser Lys Thr Gly GlnSer 145 150 155 160 Val Asp Glu Leu Phe Gln Lys Val Ala Glu Asp Tyr ValSer Val Ala 165 170 175 Ala Phe Gln Val Met Thr Glu Asp Lys Gly Val AspLeu Gly Gln Lys 180 185 190 Pro Asn Pro Tyr Phe Tyr Ser Cys Cys His His195 200 1334 base pairs nucleic acid single linear NGANNOT01 2312652 2CCCACGCGTC CGCGCGGCGC TGCGGGTAGG AGCCGGGTTG CGGGAGACCC CAGGTTCGGT 60TGGGATTCCC AGCCAGAACG GAGCTTAAGC CGGGCAGGCG AGCGAATGAC GGAGTAGCGA 120GCTGCACGGC GGCGTGCTGC GCTGTTGAGG ACGCTGTCCC GCGCGCTCCC AGGCCGCCCC 180GAGGCTTGGG GTCTTCGAAG GATAATCGGC GCCCGGGGCC GAACAGCGGG GGCACACGGG 240GCGCTGCCGA AGTGCAAGGC CACGGCCAGA GCTCGAGCCC GACGCGCTGT CTGGAGTCGT 300AGGTTGGCGC CGTTTGGGGT CGGGGTCTGA GGCTTGGGCG CTGCCTGGGC CGAGCGGAGA 360TCGGGGTTTG CCTCCCGTCC CCGCTCAGGA CCCTGACGTG GCTGAAGCGG CCCCGGGAGC 420ATGAGCGGGC AGCGCGTGGA CGTCAAGGTG GTGATGCTGG GCAAGGAGTA CGTGGGCAAG 480ACTAGCCTGG TGGAGCGCTA CGTGCACGNC NGCTTTCTGG TGGGGCCTTA TCAGAACACC 540ATCGGGGCCG CGTTCGTGGC CAAGGTGATG TCGGTCGGAG ACCGGACTGT GACATTAGGT 600ATTTGGGACA CAGCAGGCTC TGAGCGCTAT GAGGCCATGA GTAGAATCTA CTATCGGGGT 660GCCAAGGCTG CCATCGTCTG CTATGACCTC ACAGACAGCA GCAGCTTTGA GCGAGCAAAG 720TTCTGGGTGA AGGAACTGCG CAGCCTAGAG GAGGGCTGCC AAATCTACTT ATGTGGCACC 780AAGAGTGACC TGCTGGAAGA AGACCGGAGG CGTCGACGTG TGGACTTCCA CGACGTCCAG 840GACTATGCAG ACAATATCAA AGCTCAGCTC TTTGAAACAT CCAGCAAGAC AGGCCAGAGT 900GTGGACGAGC TCTTCCAGAA AGTGGCAGAG GATTACGTCA GTGTGGCTGC CTTCCAGGTG 960ATGACAGAGG ACAAGGGCGT GGATCTGGGC CAGAAGCCAA ACCCCTACTT CTACAGCTGT 1020TGTCATCACT GAGTCAGCAC TCACCTGGCC TGGGGGAATT AAAGGAATTC CCCGTAAGGG 1080CTGGACCCAG CTCCTTTCTG GGCTTGGGTA GTCAAATGTC TGAGCTACCC CAGGTCCTCA 1140TGTCAGCAGA GTGGCGCCTG CCTGTGCTGG CCCATGGAAC GGAGACAGCA TTGGGCTGAC 1200TGTGGGCATG AGGAGGGATA AGGCTGATTT GGACCCCAGG CTTCTGCCCT GGACAGCACT 1260TGTGTCTGCA GATTATTTAA GTGGCTTTTG ATCTGTAAAT AAAATCAGTG CACTGTGAAT 1320CACAAAAAAA AAGG 1334 201 amino acids amino acid single linear LIVRTUT042514506 3 Met Asn Pro Glu Tyr Asp Tyr Leu Phe Lys Leu Leu Leu Ile GlyAsp 1 5 10 15 Ser Gly Val Gly Lys Ser Cys Leu Leu Leu Arg Phe Ala AspAsp Thr 20 25 30 Tyr Thr Glu Ser Tyr Ile Ser Thr Ile Gly Val Asp Phe LysIle Arg 35 40 45 Thr Ile Glu Leu Asp Gly Lys Thr Ile Lys Leu Gln Ile TrpAsp Thr 50 55 60 Ala Gly Gln Glu Arg Phe Arg Thr Ile Thr Ser Ser Tyr TyrArg Gly 65 70 75 80 Ala His Gly Ile Ile Val Val Tyr Asp Val Thr Asp GlnGlu Ser Tyr 85 90 95 Ala Asn Val Lys Gln Trp Leu Gln Glu Ile Asp Arg TyrAla Ser Glu 100 105 110 Asn Val Asn Lys Leu Leu Val Gly Asn Lys Ser AspLeu Thr Thr Lys 115 120 125 Lys Val Val Asp Asn Thr Thr Ala Lys Glu PheAla Asp Ser Leu Gly 130 135 140 Ile Pro Phe Leu Glu Thr Ser Ala Lys AsnAla Thr Asn Val Glu Gln 145 150 155 160 Ala Phe Met Thr Met Ala Ala GluIle Lys Lys Arg Met Gly Pro Gly 165 170 175 Ala Ala Ser Gly Gly Glu ArgPro Asn Leu Lys Ile Asp Ser Thr Pro 180 185 190 Val Lys Pro Ala Gly GlyGly Cys Cys 195 200 925 base pairs nucleic acid single linear LIVRTUT042514506 4 ACCATCTTGG AACGGGAGGC GGACAGAGTC GACTGGGAGC GACCGAGCGGGCCGCCGCCG 60 CCGCCATGAA CCCCGAATAT GACTACCTGT TTAAGCTGCT TTTGATTGGCGACTCAGGCG 120 TGGGCAAGTC ATGCCTGCTC CTGCGGTTTG CTGATGACAC GTACACAGAGAGCTACATCA 180 GCACCATCGG GGTGGACTTC AAGATCCGAA CCATCGAGCT GGATGGCAAAACTATCAAAC 240 TTCAGATCTG GGACACAGCG GGCCAGGAAC GGTTCCGGAC CATCACTTCCAGCTACTACC 300 GGGGGGCTCA TGGCATCATC GTGGTGTATG ACGTCACTGA CCAGGAATCCTACGCCAACG 360 TGAAGCAGTG GCTGCAGGAG ATTGACCGCT ATGCCAGCGA GAACGTCAATAAGCTCCTGG 420 TGGGCAACAA GAGCGACCTC ACCACCAAGA AGGTGGTGGA CAACACCACAGCCAAGGAGT 480 TTGCAGACTC TCTGGGCATC CCCTTCTTGG AGACGAGCGC CAAGAATGCCACCAATGTCG 540 AGCAGGCGTT CATGACCATG GCTGCTGAAA TCAAAAAGCG GATGGGGCCTGGAGCAGCCT 600 CTGGGGGCGA GCGGCCCAAT CTCAAGATCG ACAGCACCCC TGTAAAGCCGGCTGGCGGTG 660 GCTGTTGCTA GGAGGGGCAC ATGGAGTGGG ACAGGAGGGG GCACCTTCTCCAGATGATGT 720 CCCTGGAGGG GGCAGGAGGT ACCTCCCTCT CCCTCTCCTG GGGCATTTGAGTCTGTGGCT 780 TTGGGGTGTC CTGGGCTCCC CATCTCCTTC TGGCCCATCT GCCTGCTGCCCTGAGCCCCG 840 GTTCTGTCAG GGTCCCTAAA GGAGGACACT CAGGGCCTGT GGCCAGGCAGGGCGGAAGCC 900 TGCTGTGCTG TTGCCTCTAG GTGAC 925 221 amino acids aminoacid single linear UTRSNOT16 3400003 5 Met Ser Asp Ser Glu Glu Glu SerGln Asp Arg Gln Leu Lys Ile Val 1 5 10 15 Val Leu Gly Asp Gly Ala SerGly Lys Thr Ser Leu Thr Thr Cys Phe 20 25 30 Ala Gln Glu Thr Phe Gly LysGln Tyr Lys Gln Thr Ile Gly Leu Asp 35 40 45 Phe Phe Leu Arg Arg Ile ThrLeu Pro Gly Asn Leu Asn Val Thr Leu 50 55 60 Gln Ile Trp Asp Ile Gly GlyGln Thr Ile Gly Gly Lys Met Leu Asp 65 70 75 80 Lys Tyr Ile Tyr Gly AlaGln Gly Val Leu Leu Val Tyr Asp Ile Thr 85 90 95 Asn Tyr Gln Ser Phe GluAsn Leu Glu Asp Trp Tyr Thr Val Val Lys 100 105 110 Lys Val Ser Glu GluSer Glu Thr Gln Pro Leu Val Ala Leu Val Gly 115 120 125 Asn Lys Ile AspLeu Glu His Met Arg Thr Ile Lys Pro Glu Lys His 130 135 140 Leu Arg PheCys Gln Glu Asn Gly Phe Asn Ser His Phe Val Ser Ala 145 150 155 160 LysAla Arg Asp Ser Val Phe Leu Cys Phe Gln Lys Val Val Ala Glu 165 170 175Ile Leu Gly Ile Lys Leu Asn Lys Ala Glu Ile Glu Gln Ser Gln Arg 180 185190 Val Val Lys Ala Asp Ile Val Asn Tyr Asn Gln Glu Pro Met Ala Arg 195200 205 Ala Val Asn Pro Ser Arg Ser Ser Val Cys Ala Val Gly 210 215 2202713 base pairs nucleic acid single linear 6 GGGAGGTGGG CAAGATGGCGCTTGCCGAGT GATTCTCCTC GAATACCTCC TGCCGGCGCG 60 GAGACACCGG GGCGGGGGTCCTGCCGCAAC TACCTCCCTT CCTCCTCTCC CCCGCCCCCG 120 GAGCCTTCAT CCTTCCCTTCCCCCCCCACC TCGAGGGGCG GGCCTGGTTC CCGGGACACC 180 ATGTCGGACT CTGAGGAGGAGAGCCAGGAC CGGCAACTGA AAATCGTCGT GCTGGGGGAC 240 GGCGCCTCCG GGAAGACCTCCTTAACTACG TGTTTTGCTC AAGAAACTTT TGGGAAACAG 300 TACAAACAAA CTATAGGACTGGATTTCTTT TTGAGAAGGA TAACATTGCC AGGAAACTTG 360 AATGTTACCC TTCAAATTTGGGATATAGGA GGGCAGACAA TAGGAGGCAA AATGTTGGAT 420 AAATATATCT ATGGAGCACAGGGAGTCCTC TTGGTATATG ATATTACAAA TTATCAAAGC 480 TTTGAGAATT TAGAAGATTGGTATACTGTG GTGAAGAAAG TGAGCGAGGA GTCAGAAACT 540 CAGCCACTGG TTGCCTTGGTAGGCAATAAA ATTGATTTGG AGCATATGCG AACAATAAAA 600 CCTGAAAAAC ACTTACGGTTTTGCCAGGAA AATGGTTTTA ATAGCCACTT TGTTTCAGCC 660 AAGGCAAGAG ACTCTGTCTTCCTGTGTTTT CAAAAAGTTG TTGCTGAAAT CCTTGGAATC 720 AAATTAAACA AAGCAGAAATAGAACAGTCA CAGAGGGTGG TAAAGGCAGA TATTGTAAAC 780 TACAACCAGG AACCTATGGCAAGAGCTGTT AACCCTTCTA GAAGCTCTGT GTGTGCAGTT 840 GGGTGAGCTC ATTTTTCCATTGTGTTGATA GTTTTGGCTG CCCTTCACCT CTGGGTGTGT 900 CTGAGAACTT CTAAGAACTTGTTTTATCAG TGACCATCTC TGTAGTTCAG TTAACACTTT 960 CCTCCGAACT TGCTTCATCTTTAAGTGTTC CTCCCAACCG CAGGCATGTA CTTGGGTTCA 1020 AAAGAATTCA ACTTTGGGACCACACACTTT GCATTCAAAC TGGAAGTCTC ATTCTCTGGA 1080 ATTAGACTGT TTCATTGAAAAAGAATGGTG TCCGGCCAGG CGCGGTGGCT CATGCCTGTA 1140 ATCCCAGCAC TTTGGGAGGCCGAGGCGGGT GGATCACCTG AGGTCAGGAG TTCGAGACCA 1200 GCCTGGCCAA CATGGTGAAACCCCTGTCTC TACTAAAAAT ACAAAAAAAT TAGCTGGGCG 1260 CGGTGGCGCA TGCCTGTAATCCCAGCTACT CAGGAGGCTG AGGCAGGAGA ATCACTTGAA 1320 CCCGGGAGGC AGAGGTTGCAGTGAGCCGAG ATCATGCCAT TGCACTCCAG CCTGGGTGAC 1380 AGAGCGAGAC TCCATCTCAAAAAAAATAAA TAAATAAATT ATGAATGAGT ATTTTCTAGA 1440 AATTCAACTT GCTAAGCCTGTAATACTTAA GGGTAGTTTA TCTAGATACA GTACTTTCTT 1500 CCCTGATAAG TAGTATCATTGGAGCCCTTA GGTATAGGAG AAGAGGAAGA AGTTTAAAAA 1560 GTGTAAGTGG GCCGGGCGTGGTGGCTCATG CCTGTAATCC CAGCACTTTG GGAGGCCGAG 1620 GCGGGCAGAT CACAAGGTCAGGAGATCGAG ACCATCCTGG CCAAGATGGT GAAACCCCGT 1680 CTCTACTAAA AATACAAAATTAGCCGGCCG TGGTGGCTCA CACCTGTAAT CCCAGCTACT 1740 TGGGAGGCTG AGGCAGGAGAATCACTTGAG GTCAGGAGTT TGAGACCAGC CTGGCCAACG 1800 TGGTGAAACC CTGTCTCTACTAAAAATACA AAAACTAGGC CAGGCGCGGG TGGCACGCCT 1860 GTAATCTCAG CACTTTGGGAGGCCGAGGCA GGTGGATCAC CTGAGGTCAG GAGTTCAAGA 1920 CCAGCCTGGC CAACATGGTGAAACCCCACC TCTACTAAAA ATACAAAAAT TAGCCAGGCA 1980 TGGTGGTGCA TGCCTGTAATCCCAGCTACT TGGGAGGCTG AGGCAGGAGA ATTGCTTGAA 2040 CCCGGGAGCA GAGGTTGCAGTGAGCCGAGA TCATGCCACT GCACTCCAGC CTGGGTGACA 2100 GAGCGAGACT CCATTTCAAAAAAAAGAACT ACAAGTTCTG ATTCCGGACT CCCAGATGTG 2160 AGTTTTAATC TCCTCTCCACTGATTGATCC TGACTAATCA CTAGCCCCCT GTGCCCAATT 2220 TCAACAGTAT GCTGGAGTCAAATCTGAACC CCAAACTATG CCCTCTTAAG GGGGGTCCCT 2280 CTGGGATGCC AACATGCATTCACTTCTTCA CCTGGCTAGG CATTCCATGA GTATTCACAT 2340 TGTAGTCACT CCCCTAGGGCTATGCCCAGG AGTTAGTACT TTCCTACCAC TTGGTGATCT 2400 TGAGTGAGTT TTGGATGTCCTCAATGGGTC CTGAGATGAG TCAGAGGAGA GCTAGAGTTG 2460 GGAACTGATC ACCAGTGGCCCCCCCAGTCC TCAGCTCTTG AAGGAAAGGG AATGAATTGC 2520 TCTGGCCATT TGCATCTGTGCGAAGGATCG AACAAAGCCA CTTTCTACAA TGCAACCCTG 2580 TCCGACGGCC CCCTTCCCAAAGCTGCCTGC AACTTTCAAC CCCGNTGAAT GGACTTTGGA 2640 ACTTGGGACA GAGGCAAAGACTTAAATGAG GNGCCAAAGN AATGTCGGGT CCATTACCAA 2700 ATTAGGAGGG TNG 2713 203amino acids amino acid single linear GenBank 438164 7 Met Ser Gly GlnArg Val Asp Val Lys Val Val Met Leu Gly Lys Glu 1 5 10 15 Tyr Val GlyLys Thr Ser Leu Val Glu Arg Tyr Val His Asp Arg Phe 20 25 30 Leu Val GlyPro Tyr Gln Asn Thr Ile Gly Ala Ala Phe Val Ala Lys 35 40 45 Val Met CysVal Gly Asp Arg Thr Val Thr Leu Gly Ile Trp Asp Thr 50 55 60 Ala Gly SerGlu Arg Tyr Glu Ala Met Ser Arg Ile Tyr Tyr Arg Gly 65 70 75 80 Ala LysAla Ala Ile Val Cys Tyr Asp Leu Thr Asp Ser Ser Ser Phe 85 90 95 Glu ArgAla Lys Phe Trp Val Lys Glu Leu Arg Ser Leu Glu Glu Gly 100 105 110 CysGln Ile Tyr Leu Cys Gly Thr Lys Ser Asp Leu Leu Glu Glu Asp 115 120 125Arg Arg Arg Arg Arg Val Asp Phe His Asp Val Gln Asp Tyr Ala Asp 130 135140 Asn Ile Lys Ala Gln Leu Phe Glu Thr Ser Ser Lys Thr Gly Gln Ser 145150 155 160 Val Asp Glu Leu Phe Gln Lys Val Ala Glu Asp Tyr Val Ser ValAla 165 170 175 Ala Phe Gln Val Met Thr Glu Asp Lys Gly Val Asp Leu SerGln Lys 180 185 190 Ala Asn Pro Tyr Phe Tyr Ser Cys Cys His His 195 200201 amino acids amino acid single linear GenBank 57006 8 Met Asn Pro GluTyr Asp Tyr Leu Phe Lys Leu Leu Leu Ile Gly Asp 1 5 10 15 Ser Gly ValGly Lys Ser Cys Leu Leu Leu Arg Phe Ala Asp Asp Thr 20 25 30 Tyr Thr GluSer Tyr Ile Ser Thr Ile Gly Val Asp Phe Lys Ile Arg 35 40 45 Thr Ile GluLeu Asp Gly Lys Thr Ile Lys Leu Gln Ile Trp Asp Thr 50 55 60 Ala Gly GlnGlu Arg Phe Arg Thr Val Thr Ser Ser Tyr Tyr Arg Gly 65 70 75 80 Ala HisGly Ile Ile Val Val Tyr Asp Val Thr Asp Gln Glu Ser Tyr 85 90 95 Ala AsnVal Lys Gln Trp Leu Gln Glu Ile Asp Arg Tyr Ala Ser Glu 100 105 110 AsnVal Asn Lys Leu Leu Val Gly Asn Lys Ser Asp Leu Thr Thr Lys 115 120 125Lys Val Val Asp Asn Thr Thr Ala Lys Glu Phe Ala Asp Ser Leu Gly 130 135140 Val Pro Phe Leu Glu Thr Ser Ala Lys Asn Ala Thr Asn Val Glu Gln 145150 155 160 Ala Phe Met Thr Met Ala Ala Glu Ile Lys Lys Arg Met Gly ProGly 165 170 175 Ala Ala Ser Gly Gly Glu Arg Pro Asn Leu Lys Ile Asp SerThr Pro 180 185 190 Val Lys Ser Ala Ser Gly Gly Cys Cys 195 200 221amino acids amino acid single linear GenBank 1154901 9 Met Ser Asp SerGlu Glu Glu Ser Gln Asp Arg Gln Leu Lys Ile Val 1 5 10 15 Val Leu GlyAsp Gly Thr Ser Gly Lys Thr Ser Leu Ala Thr Cys Phe 20 25 30 Ala Gln GluThr Phe Gly Lys Gln Tyr Lys Gln Thr Ile Gly Leu Asp 35 40 45 Phe Phe LeuArg Arg Ile Thr Leu Pro Gly Asn Leu Asn Val Thr Leu 50 55 60 Gln Val TrpAsp Ile Gly Gly Gln Thr Ile Gly Gly Lys Met Leu Asp 65 70 75 80 Lys TyrIle Tyr Gly Ala Gln Gly Ile Leu Leu Val Tyr Asp Ile Thr 85 90 95 Asn TyrGln Ser Phe Glu Asn Leu Glu Asp Trp Tyr Ser Val Val Lys 100 105 110 ThrVal Ser Glu Glu Ser Glu Thr Gln Pro Leu Val Ala Leu Val Gly 115 120 125Asn Lys Ile Asp Leu Glu His Met Arg Thr Val Lys Pro Asp Lys His 130 135140 Leu Arg Phe Cys Gln Glu Asn Gly Phe Ser Ser His Phe Val Ser Ala 145150 155 160 Lys Thr Gly Asp Ser Val Phe Leu Cys Phe Gln Lys Val Ala AlaGlu 165 170 175 Ile Leu Gly Ile Lys Leu Asn Lys Ala Glu Ile Glu Gln SerGln Arg 180 185 190 Val Val Lys Ala Asp Ile Val Asn Tyr Asn Gln Glu ProMet Ser Arg 195 200 205 Thr Val Asn Pro Pro Arg Ser Ser Met Cys Ala ValGln 210 215 220

What is claimed is:
 1. An isolated polypeptide selected from the groupconsisting of: a) a polypeptide comprising an amino acid sequenceselected from the group consisting of SEQ ID NO:1, SEQ ID NO:3, and SEQID NO:5, b) a polypeptide comprising a naturally occurring amino acidsequence at least 90% identical to an amino acid sequence selected fromthe group consisting of SEQ ID NO:1, SEQ ID NO:3, and SEQ ID NO:5, c) abiologically active fragment of a polypeptide having an amino acidsequence selected from the group consisting of SEQ ID NO:1, SEQ ID NO:3,and SEQ ID NO:5, and d) an immunogenic fragment of a polypeptide havingan amino acid sequence selected from the group consisting of SEQ IDNO:1, SEQ ID NO:3, and SEQ ID NO:5.
 2. An isolated polypeptide of claim1 selected from the group consisting of SEQ ID NO:1, SEQ ID NO:3, andSEQ ID NO:5.
 3. An isolated polynucleotide encoding a polypeptide ofclaim
 1. 4. An isolated polynucleotide encoding a polypeptide of claim2.
 5. An isolated polynucleotide of claim 4 selected from the groupconsisting of SEQ ID NO:2, SEQ ID NO:4, and SEQ ID NO:6.
 6. Arecombinant polynucleotide comprising a promoter sequence operablylinked to a polynucleotide of claim
 3. 7. A cell transformed with arecombinant polynucleotide of claim
 6. 8. A transgenic organismcomprising a recombinant polynucleotide of claim
 6. 9. A method ofproducing a polypeptide of claim 1, the method comprising: a) culturinga cell under conditions suitable for expression of the polypeptide,wherein said cell is transformed with a recombinant polynucleotide, andsaid recombinant polynucleotide comprises a promoter sequence operablylinked to a polynucleotide encoding the polypeptide of claim 1, and b)recovering the polypeptide so expressed.
 10. A method of claim 9,wherein the polypeptide has an amino acid sequence selected from thegroup consisting of SEQ ID NO:1, SEQ ID NO:3, and SEQ ID NO:5.
 11. Anisolated antibody which specifically binds to a polypeptide of claim 1.12. An isolated polynucleotide selected from the group consisting of: a)a polynucleotide comprising a polynucleotide sequence selected from thegroup consisting of SEQ ID NO:2, SEQ ID NO:4, and SEQ ID NO:6, b) apolynucleotide comprising a naturally occurring polynucleotide sequenceat least 90% identical to a polynucleotide sequence selected from thegroup consisting of SEQ ID NO:2, SEQ ID NO:4, and SEQ ID NO:6, c) apolynucleotide complementary to a polynucleotide of a), d) apolynucleotide complementary to a polynucleotide of b), and e) an RNAequivalent of a)-d).
 13. An isolated polynucleotide comprising at least60 contiguous nucleotides of a polynucleotide of claim
 12. 14. A methodof detecting a target polynucleotide in a sample, said targetpolynucleotide having a sequence of a polynucleotide of claim 12, themethod comprising: a) hybridizing the sample with a probe comprising atleast 20 contiguous nucleotides comprising a sequence complementary tosaid target polynucleotide in the sample, and which probe specificallyhybridizes to said target polynucleotide, under conditions whereby ahybridization complex is formed between said probe and said targetpolynucleotide or fragments thereof, and b) detecting the presence orabsence of said hybridization complex, and, optionally, if present, theamount thereof.
 15. A method of claim 14, wherein the probe comprises atleast 60 contiguous nucleotides.
 16. A method of detecting a targetpolynucleotide in a sample, said target polynucleotide having a sequenceof a polynucleotide of claim 12, the method comprising: a) amplifyingsaid target polynucleotide or fragment thereof using polymerase chainreaction amplification, and b) detecting the presence or absence of saidamplified target polynucleotide or fragment thereof, and, optionally, ifpresent, the amount thereof.
 17. A composition comprising a polypeptideof claim 1 and a pharmaceutically acceptable excipient.
 18. Acomposition of claim 17, wherein the polypeptide has an amino acidsequence selected from the group consisting of SEQ ID NO:1, SEQ ID NO:3,and SEQ ID NO:5.
 19. A method for treating a disease or conditionassociated with decreased expression of functional RABP, comprisingadministering to a patient in need of such treatment the composition ofclaim
 17. 20. A method of screening a compound for effectiveness as anagonist of a polypeptide of claim 1, the method comprising: a) exposinga sample comprising a polypeptide of claim 1 to a compound, and b)detecting agonist activity in the sample.
 21. A composition comprisingan agonist compound identified by a method of claim 20 and apharmaceutically acceptable excipient.
 22. A method for treating adisease or condition associated with decreased expression of functionalRABP, comprising administering to a patient in need of such treatment acomposition of claim
 21. 23. A method of screening a compound foreffectiveness as an antagonist of a polypeptide of claim 1, the methodcomprising: a) exposing a sample comprising a polypeptide of claim 1 toa compound, and b) detecting antagonist activity in the sample.
 24. Acomposition comprising an antagonist compound identified by a method ofclaim 23 and a pharmaceutically acceptable excipient.
 25. A method fortreating a disease or condition associated with overexpression offunctional RABP, comprising administering to a patient in need of suchtreatment a composition of claim
 24. 26. A method of screening for acompound that specifically binds to the polypeptide of claim 1, themethod comprising: a) combining the polypeptide of claim 1 with at leastone test compound under suitable conditions, and b) detecting binding ofthe polypeptide of claim 1 to the test compound, thereby identifying acompound that specifically binds to the polypeptide of claim
 1. 27. Amethod of screening for a compound that modulates the activity of thepolypeptide of claim 1, the method comprising: a) combining thepolypeptide of claim 1 with at least one test compound under conditionspermissive for the activity of the polypeptide of claim 1, b) assessingthe activity of the polypeptide of claim 1 in the presence of the testcompound, and c) comparing the activity of the polypeptide of claim 1 inthe presence of the test compound with the activity of the polypeptideof claim 1 in the absence of the test compound, wherein a change in theactivity of the polypeptide of claim 1 in the presence of the testcompound is indicative of a compound that modulates the activity of thepolypeptide of claim
 1. 28. A method of screening a compound foreffectiveness in altering expression of a target polynucleotide, whereinsaid target polynucleotide comprises a sequence of claim 5, the methodcomprising: a) exposing a sample comprising the target polynucleotide toa compound, under conditions suitable for the expression of the targetpolynucleotide, b) detecting altered expression of the targetpolynucleotide, and c) comparing the expression of the targetpolynucleotide in the presence of varying amounts of the compound and inthe absence of the compound.
 29. A method of assessing toxicity of atest compound, the method comprising: a) treating a biological samplecontaining nucleic acids with the test compound, b) hybridizing thenucleic acids of the treated biological sample with a probe comprisingat least 20 contiguous nucleotides of a polynucleotide of claim 12 underconditions whereby a specific hybridization complex is formed betweensaid probe and a target polynucleotide in the biological sample, saidtarget polynucleotide comprising a polynucleotide sequence of apolynucleotide of claim 12 or fragment thereof, c) quantifying theamount of hybridization complex, and d) comparing the amount ofhybridization complex in the treated biological sample with the amountof hybridization complex in an untreated biological sample, wherein adifference in the amount of hybridization complex in the treatedbiological sample is indicative of toxicity of the test compound.
 30. Adiagnostic test for a condition or disease associated with theexpression of RABP in a biological sample, the method comprising: a)combining the biological sample with an antibody of claim 11, underconditions suitable for the antibody to bind the polypeptide and form anantibody:polypeptide complex, and b) detecting the complex, wherein thepresence of the complex correlates with the presence of the polypeptidein the biological sample.
 31. The antibody of claim 11, wherein theantibody is: a) a chimeric antibody, b) a single chain antibody, c) aFab fragment, d) a F(ab′)₂ fragment, or e) a humanized antibody.
 32. Acomposition comprising an antibody of claim 11 and an acceptableexcipient.
 33. A method of diagnosing a condition or disease associatedwith the expression of RABP in a subject, comprising administering tosaid subject an effective amount of the composition of claim
 32. 34. Acomposition of claim 32, wherein the antibody is labeled.
 35. A methodof diagnosing a condition or disease associated with the expression ofRABP in a subject, comprising administering to said subject an effectiveamount of the composition of claim
 34. 36. A method of preparing apolyclonal antibody with the specificity of the antibody of claim 11,the method comprising: a) immunizing an animal with a polypeptide havingan amino acid sequence selected from the group consisting of SEQ IDNO:1, SEQ ID NO:3, and SEQ ID NO:5, or an immunogenic fragment thereof,under conditions to elicit an antibody response, b) isolating antibodiesfrom said animal, and c) screening the isolated antibodies with thepolypeptide, thereby identifying a polyclonal antibody which bindsspecifically to a polypeptide having an amino acid sequence selectedfrom the group consisting of SEQ ID NO:1, SEQ ID NO:3, and SEQ ID NO:5.37. A polyclonal antibody produced by a method of claim
 36. 38. Acomposition comprising the polyclonal antibody of claim 37 and asuitable carrier.
 39. A method of making a monoclonal antibody with thespecificity of the antibody of claim 11, the method comprising: a)immunizing an animal with a polypeptide having an amino acid sequenceselected from the group consisting of SEQ ID NO:1, SEQ ID NO:3, and SEQID NO:5, or an immunogenic fragment thereof, under conditions to elicitan antibody response, b) isolating antibody producing cells from theanimal, c) fusing the antibody producing cells with immortalized cellsto form monoclonal antibody-producing hybridoma cells, d) culturing thehybridoma cells, and e) isolating from the culture monoclonal antibodywhich binds specifically to a polypeptide having an amino acid sequenceselected from the group consisting of SEQ ID NO:1, SEQ ID NO:3, and SEQID NO:5.
 40. A monoclonal antibody produced by a method of claim
 39. 41.A composition comprising the monoclonal antibody of claim 40 and asuitable carrier.
 42. The antibody of claim 11, wherein the antibody isproduced by screening a Fab expression library.
 43. The antibody ofclaim 11, wherein the antibody is produced by screening a recombinantimmunoglobulin library.
 44. A method of detecting a polypeptide havingan amino acid sequence selected from the group consisting of SEQ IDNO:1, SEQ ID NO:3, and SEQ ID NO:5 in a sample, the method comprising:a) incubating the antibody of claim 11 with a sample under conditions toallow specific binding of the antibody and the polypeptide, and b)detecting specific binding, wherein specific binding indicates thepresence of a polypeptide having an amino acid sequence selected fromthe group consisting of SEQ ID NO:1, SEQ ID NO:3, and SEQ ID NO:5 in thesample.
 45. A method of purifying a polypeptide having an amino acidsequence selected from the group consisting of SEQ ID NO:1, SEQ ID NO:3,and SEQ ID NO:5 from a sample, the method comprising: a) incubating theantibody of claim 11 with a sample under conditions to allow specificbinding of the antibody and the polypeptide, and b) separating theantibody from the sample and obtaining the purified polypeptide havingan amino acid sequence selected from the group consisting of SEQ IDNO:1, SEQ ID NO:3, and SEQ ID NO:5.
 46. A microarray wherein at leastone element of the microarray is a polynucleotide of claim
 13. 47. Amethod of generating a transcript image of a sample which containspolynucleotides, the method comprising: a) labeling the polynucleotidesof the sample, b) contacting the elements of the microarray of claim 46with the labeled polynucleotides of the sample under conditions suitablefor the formation of a hybridization complex, and c) quantifying theexpression of the polynucleotides in the sample.
 48. An array comprisingdifferent nucleotide molecules affixed in distinct physical locations ona solid substrate, wherein at least one of said nucleotide moleculescomprises a first oligonucleotide or polynucleotide sequencespecifically hybridizable with at least 30 contiguous nucleotides of atarget polynucleotide, and wherein said target polynucleotide is apolynucleotide of claim
 12. 49. An array of claim 48, wherein said firstoligonucleotide or polynucleotide sequence is completely complementaryto at least 30 contiguous nucleotides of said target polynucleotide. 50.An array of claim 48, wherein said first oligonucleotide orpolynucleotide sequence is completely complementary to at least 60contiguous nucleotides of said target polynucleotide.
 51. An array ofclaim 48, wherein said first oligonucleotide or polynucleotide sequenceis completely complementary to said target polynucleotide.
 52. An arrayof claim 48, which is a microarray.
 53. An array of claim 48, furthercomprising said target polynucleotide hybridized to a nucleotidemolecule comprising said first oligonucleotide or polynucleotidesequence.
 54. An array of claim 48, wherein a linker joins at least oneof said nucleotide molecules to said solid substrate.
 55. An array ofclaim 48, wherein each distinct physical location on the substratecontains multiple nucleotide molecules, and the multiple nucleotidemolecules at any single distinct physical location have the samesequence, and each distinct physical location on the substrate containsnucleotide molecules having a sequence which differs from the sequenceof nucleotide molecules at another distinct physical location on thesubstrate.
 56. A polypeptide of claim 1, comprising the amino acidsequence of SEQ ID NO:1.
 57. A polypeptide of claim 1, comprising theamino acid sequence of SEQ ID NO:3.
 58. A polypeptide of claim 1,comprising the amino acid sequence of SEQ ID NO:5.
 59. A polynucleotideof claim 12, comprising the polynucleotide sequence of SEQ ID NO:2. 60.A polynucleotide of claim 12, comprising the polynucleotide sequence ofSEQ ID NO:4.
 61. A polynucleotide of claim 12, comprising thepolynucleotide sequence of SEQ ID NO:6.